Antimicrobial Peptide Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to variants of a parent antimicrobial peptide. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to variants of an antimicrobial peptide, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.

2. Description of the Related Art

Several classes of antimicrobial peptides (AMPs) have been described in literature, examples of which include defensins and alpha-helical peptides.

The present invention provides variants of an antimicrobial peptide isolated from Arenicola marina, and described in WO 2007/023163.

The variant antimicrobial peptides of the present invention exhibit improved antimicrobial activity as compared to the parent antimicrobial peptide. In particular, the variants exhibit improved antimicrobial activity in the presence of serum and blood proteins. Another advantage of the variant peptides of the invention is a reduced protein binding e.g. to serum and blood proteins, which results in an improved bioavailability as compared to the parent antimicrobial peptide.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising an alteration at one or more (several) of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity.

The present invention also relates to isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants.

The present invention also relates to a method of treating a microbial infection using the variants of the invention; and use of variants for manufacturing a medicament for the treatment of a microbial infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising an alteration at one or more (several) of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity.

DEFINITIONS

Antimicrobial activity: The term “antimicrobial activity” is defined herein as an activity which is capable of killing or inhibiting growth of microbial cells. In the context of the present invention the term “antimicrobial” is intended to mean that there is a bactericidal and/or a bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal effect, wherein the term “bactericidal” is to be understood as capable of killing bacterial cells. The term “bacteriostatic” is to be understood as capable of inhibiting bacterial growth, i.e. inhibiting growing bacterial cells. The term “fungicidal” is to be understood as capable of killing fungal cells. The term “fungistatic” is to be understood as capable of inhibiting fungal growth, i.e. inhibiting growing fungal cells. The term “virucidal” is to be understood as capable of inactivating virus. The term “microbial cells” denotes bacterial or fungal cells (including yeasts).

In the context of the present invention the term “inhibiting growth of microbial cells” is intended to mean that the cells are in the non-growing state, i.e., that they are not able to propagate.

In a preferred embodiment, the term “antimicrobial activity” is defined as bactericidal and/or bacteriostatic activity. More preferably, “antimicrobial activity” is defined as bactericidal and/or bacteriostatic activity against Escherichia, preferably Escherichia coli.

For purposes of the present invention, antimicrobial activity may be determined according to the procedure described by Lehrer et al., 1991, Journal of Immunological Methods 137(2): 167-174. Alternatively, antimicrobial activity may be determined according to the NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards).

Peptides having antimicrobial activity may be capable of reducing the number of living cells of Escherichia coli (DSM 1576) to 1/100 after 8 hours (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and in particular after 30 minutes) incubation at 37° C. in a relevant microbial growth substrate at a concentration of 500 micrograms/ml; preferably at a concentration of 250 micrograms/ml; more preferably at a concentration of 100 micrograms/ml; even more preferably at a concentration of 50 micrograms/ml; most preferably at a concentration of 25 micrograms/ml; and in particular at a concentration of 10 micrograms/ml of the peptides having antimicrobial activity.

Peptides having antimicrobial activity may also be capable of inhibiting the outgrowth of Escherichia coli (DSM 1576) for 8 hours at 37° C. in a relevant microbial growth substrate, when added in a concentration of 500 micrograms/ml; preferably when added in a concentration of 250 micrograms/ml; more preferably when added in a concentration of 100 micrograms/ml; even more preferably when added in a concentration of 50 micrograms/ml; most preferably when added in a concentration of 10 micrograms/ml; and in particular when added in a concentration of 5 micrograms/ml.

The variant peptides of the present invention have improved antimicrobial activity compared to the antimicrobial peptide of SEQ ID NO: 2. In an embodiment, the variant peptides of the present invention have more than 100% of the antimicrobial activity of the peptide of SEQ ID NO: 2 in the presence of blood serum.

Variant: The term “variant” means a peptide having antimicrobial activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Wild-type antimicrobial peptide: The term “wild-type” antimicrobial peptide means an antimicrobial peptide expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

Parent or Parent antimicrobial peptide: The term “parent” or “parent antimicrobial peptide” means an antimicrobial peptide to which an alteration is made to produce the enzyme variants of the present invention. The parent may be a naturally occurring (wild-type) peptide or a variant thereof.

Isolated variant: The term “isolated variant” means a variant that is modified by the hand of man. In one aspect, the variant is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE.

Substantially pure variant: The term “substantially pure variant” means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other peptide material with which it is natively or recombinantly associated. Preferably, the variant is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total peptide material present in the preparation. The variants of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant by well known recombinant methods or by classical purification methods.

Mature peptide: The term “mature peptide” means a peptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Mature peptide coding sequence: The term “mature peptide coding sequence” means a polynucleotide that encodes a mature peptide having antimicrobial activity.

Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Fragment: The term “fragment” means a peptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of a mature peptide; wherein the fragment has antimicrobial activity. In one aspect, a fragment contains at least 15 amino acid residues, e.g., at least 17 and at least 19 amino acid residues (e.g., amino acids 1 to 20 of SEQ ID NO: 2).

Subsequence: The term “subsequence” means a polynucleotide having one or more (several) nucleotides deleted from the 5′- and/or 3′-end of a mature peptide coding sequence; wherein the subsequence encodes a fragment having antimicrobial activity.

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded peptide) or may encode peptides having altered amino acid sequences. An allelic variant of a peptide is a peptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” means a polynucleotide that is modified by the hand of man. In one aspect, the isolated polynucleotide is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose electrophoresis. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Substantially pure polynucleotide: The term “substantially pure polynucleotide” means a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered peptide production systems. Thus, a substantially pure polynucleotide contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′- and 3′-untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, e.g., at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of its peptide product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

Control sequences: The term “control sequences” means all components necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to additional nucleotides that provide for its expression.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Conventions for Designation of Variants

For purposes of the present invention, the mature peptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another antimicrobial peptide. The amino acid sequence of another antimicrobial peptide is aligned with the mature peptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature peptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.

Identification of the corresponding amino acid residue in another antimicrobial peptide can be confirmed by an alignment of multiple peptide sequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23: 2947-2948).

When the other enzyme has diverged from the mature peptide of SEQ ID NO: 2 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of peptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the peptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the peptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and implementations of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the antimicrobial peptide variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”. In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.

Different Substitutions.

Where different substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of arginine with tyrosine or glutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Parent Antimicrobial Peptides

The parent antimicrobial peptide is (a) a peptide with at least 60% sequence identity with the mature peptide of SEQ ID NO: 2; (b) a peptide encoded by a polynucleotide that hybridizes under medium stringency conditions with (i) the mature peptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary strand of (i); or (c) a peptide encoded by a polynucleotide with at least 60% sequence identity with the mature peptide coding sequence of SEQ ID NO: 1.

In a first aspect, the parent has a sequence identity to the mature peptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have antimicrobial activity. In one aspect, the amino acid sequence of the parent differs by no more than ten amino acids, e.g., by five amino acids, by four amino acids, by three amino acids, by two amino acids, and by one amino acid from the mature peptide of SEQ ID NO: 2.

The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2.

In an embodiment, the parent is a fragment of the peptide of SEQ ID NO: 2 containing at least 15 amino acid residues, e.g., at least 17 and at least 19 amino acid residues.

In another embodiment, the parent is an allelic variant of the peptide of SEQ ID NO: 2.

In a second aspect, the parent peptide is encoded by a polynucleotide that hybridizes under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature peptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary strand of (i) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, e.g., at least 25, or at least 35 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleotide probe corresponding to the polynucleotide shown in SEQ ID NO: 1, its complementary strand, or a subsequence thereof, under low to very high stringency conditions. Molecules to which the probe hybridizes can be detected using, for example, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is SEQ ID NO: 1.

For long probes of at least 60 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C. (low stringency), 55° C. (medium stringency), 60° C. (medium-high stringency), 65° C. (high stringency), or 70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 60 nucleotides in length, stringency conditions are defined as prehybridization and hybridization at about 5° C. to about 10° C. below the calculated T_(m) using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

In a third aspect, the parent is encoded by a polynucleotide with a sequence identity to the peptide coding sequence of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which encodes a peptide having antimicrobial activity.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a cell in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

The parent may be a bacterial antimicrobial peptide. For example, the parent may be a gram-positive bacterial peptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces antimicrobial peptide, or a gram-negative bacterial peptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma antimicrobial peptide.

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis antimicrobial peptide.

In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus antimicrobial peptide.

In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans antimicrobial peptide.

The parent may be a fungal antimicrobial peptide. For example, the parent may be a yeast antimicrobial peptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia antimicrobial peptide. For example, the parent may be a filamentous fungal antimicrobial peptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria antimicrobial peptide.

In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis antimicrobial peptide.

In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminurn, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride antimicrobial peptide.

In another aspect, the parent is an Arenicola marina antimicrobial peptide, e.g., the antimicrobial peptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding a parent may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with a probe(s), the polynucleotide may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

The parent may be a hybrid peptide in which a portion of one peptide is fused at the N-terminus or the C-terminus of a portion of another peptide.

The parent also may be a fused peptide or cleavable fusion peptide in which one peptide is fused at the N-terminus or the C-terminus of another peptide. A fused peptide is produced by fusing a polynucleotide encoding one peptide to a polynucleotide encoding another peptide. Techniques for producing fusion peptides are known in the art, and include ligating the coding sequences encoding the peptides so that they are in frame and that expression of the fused peptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion peptide can further comprise a cleavage site between the two peptides. Upon secretion of the fusion protein, the site is cleaved releasing the two peptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Preparation of Variants

The present invention also relates to methods for obtaining a variant having antimicrobial activity, comprising: (a) introducing into a parent antimicrobial peptide a substitution at one or more (several) corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the mature peptide of SEQ ID NO: 2, wherein the variant has antimicrobial activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several) mutations are created at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a peptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a peptide.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Variants

The present invention also provides variants of a parent antimicrobial peptide comprising a substitution at one or more (several) positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 (preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17), wherein the variant has antimicrobial activity. In an embodiment, the variant has improved antimicrobial activity compared to the peptide of SEQ ID NO: 2; preferably in the presence of blood or serum. In another embodiment, the variant exhibit less protein binding compared to the peptide of SEQ ID NO: 2. Preferably, the variant antimicrobial peptides exhibit at the most 99% serum protein binding. The variant antimicrobial peptides also exhibit improved bioavailability. Preferably the subcutaneous bioavailability is at least 30%, more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60%.

In an embodiment, the variant has sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to the amino acid sequence of the parent antimicrobial peptide.

In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature peptide of SEQ ID NO: 2.

In one aspect, the number of substitutions in the variants of the present invention is 1-11, e.g., 1-10 substitutions, 1-9 substitutions, 1-8 substitutions, 1-7 substitutions, 1-6 substitutions, 1-5 substitutions, 1-4 substitutions, 1-3 substitutions and 1-2 substitutions.

In one aspect, the variant comprises or consists of the amino acid sequence shown as SEQ ID NO: 3 to SEQ ID NO: 548.

The term “SEQ ID NO: 3 to SEQ ID NO: 548” is intended to mean any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 423, SEQ ID NO: 424, SEQ ID NO: 425, SEQ ID NO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507, SEQ ID NO: 508, SEQ ID NO: 509, SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516, SEQ ID NO: 517, SEQ ID NO: 518, SEQ ID NO: 519, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, and/or SEQ ID NO: 548.

In one aspect, a variant comprises a substitution at one or more (several) positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at four positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at five positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17. In another aspect, a variant comprises a substitution at six positions corresponding to any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21; preferably positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and 21; and more preferably positions 4, 5, 6, 8, 9, 12, 13, 15, and 17.

In another aspect, the variant comprises the substitution G1A,D,F,H,I,K,M,Q,R,S,T,V,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution F2A,G,H,I,L,M,P,S,V,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution C3L of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution W4A,E,F,G,I,L,M,N,Q,S,T,V,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y5E,F,G,H,K,N,R,S,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V6A,C,E,F,G,H,I,L,M,N,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution C7V of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V8A,F,G,H,I,L,N,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y9A,D,F,G,H,I,K,M,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution R10K,P,S,T of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution N11A,G,H,Q,R,S,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution G12A,D,E,F,H,K,N,R,S,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V13A,C,F,G,H,K,L,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution V15A,C,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution Y17C,F,G,H,I,K,L,M,N,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution R19D,H,K,M,T,Y of the mature peptide of SEQ ID NO: 2. In another aspect, the variant comprises the substitution N21A,C,F,G,H,I,K,L,M,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 1. In another aspect, the amino acid at position 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Phe, His, Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. In another aspect, the variant comprises the substitution G1A,D,F,H,I,K,M,Q,R,S,T,V,W,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 2. In another aspect, the amino acid at position 2 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Gly, His, Ile, Leu, Met, Pro, Ser, Val, Trp, or Tyr. In another aspect, the variant comprises the substitution F2A,G,H,I,L,M,P,S,V,W,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 3. In another aspect, the amino acid at position 3 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, the variant comprises the substitution C3L of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 4. In another aspect, the amino acid at position 4 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Glu, Phe, Gly, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val, or Tyr. In another aspect, the variant comprises the substitution W4A,E,F,G,I,L,M,N,Q,S,T,V,Y of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 5. In another aspect, the amino acid at position 5 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu, Phe, Gly, His, Lys, Asn, Arg, Ser, or Trp. In another aspect, the variant comprises the substitution Y5E,F,G,H,K,N,R,S,W of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 6. In another aspect, the amino acid at position 6 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V6A,C,E,F,G,H,I,L,M,N,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 7. In another aspect, the amino acid at position 7 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, the variant comprises the substitution C7V of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 8. In another aspect, the amino acid at position 8 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V8A,F,G,H,I,L,N,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 9. In another aspect, the amino acid at position 9 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Phe, Gly, His, Ile, Lys, Met, Gln, Arg, Ser, Thr, Val, or Trp. In another aspect, the variant comprises the substitution Y9A,D,F,G,H,I,K,M,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 10. In another aspect, the amino acid at position 10 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Lys, Pro, Ser, or Thr. In another aspect, the variant comprises the substitution R10K,P,S,T of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 11. In another aspect, the amino acid at position 11 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Gly, His, Gln, Arg, Ser, or Tyr. In another aspect, the variant comprises the substitution N11A,G,H,Q,R,S,Y of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 12. In another aspect, the amino acid at position 12 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Asp, Glu, Phe, His, Lys, Asn, Arg, Ser, or Tyr. In another aspect, the variant comprises the substitution G12A,D,E,F,H,K,N,R,S,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 13. In another aspect, the amino acid at position 13 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V13A,C,F,G,H,K,L,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at position 15. In another aspect, the amino acid at position 15 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution V15A,C,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 17. In another aspect, the amino acid at position 17 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Trp. In another aspect, the variant comprises the substitution Y17C,F,G,H,I,K,L,M,N,Q,R,S,T,V,W of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 19. In another aspect, the amino acid at position 19 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp, His, Lys, Met, Thr, or Tyr. In another aspect, the variant comprises the substitution R19D,H,K,M,T,Y of the mature peptide of SEQ ID NO: 2.

In one aspect, the variant comprises a substitution at position 21. In another aspect, the amino acid at position 21 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises the substitution N21A,C,F,G,H,I,K,L,M,P,Q,R,S,T,W,Y of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises a substitution at positions corresponding to positions 5 and 17, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5 and 9, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5 and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 17 and 9, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 17 and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 9 and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, and 9, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 9, and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 17, 9, and 15, such as those described above.

In another aspect, the variant comprises substitutions at positions corresponding to positions 5, 17, 9, and 15, such as those described above.

In another aspect, the variant comprises one or more (several) substitutions selected from the group consisting of

G1A, G1D, G1F, G1H, G1I, G1K, G1M, G1Q, G1R, G1S, G1T, G1V, G1W, G1Y, F2A, F2G, F2H, F2I, F2L, F2M, F2P, F2S, F2V, F2W, F2Y, C3L, W4A, W4E, W4F, W4G, W41, W4L, W4M, W4N, W4Q, W4S, W4T, W4V, W4Y, Y5E, Y5F, Y5G, Y5H, Y5K, Y5N, Y5R, Y5S, Y5W, V6A, V6C, V6E, V6F, V6G, V6H, V61, V6L, V6M, V6N, V6Q, V6R, V6S, V6T, V6W, V6Y, C7V, V8A, V8F, V8G, V8H, V81, V8L, V8N, V8S, V8T, V8W, V8Y, Y9A, Y9D, Y9F, Y9G, Y9H, Y91, Y9K, Y9M, Y9Q, Y9R, Y9S, Y9T, Y9V, Y9W, R10K, R10P, R10S, R10T, N11A, N11G, N11H, N11Q, N11R, N115, N11Y, G12A, G12D, G12E, G12F, G12H, G12K, G12N, G12R, G12S, G12Y, V13A, V13C, V13F, V13G, V13H, V13K, V13L, V13P, V13Q, V13R, V13S, V13T, V13W, V13Y, V15A, V15C, V15F, V15G, V15H, V151, V15K, V15L, V15M, V15N, V15P, V15Q, V15R, V15S, V15T, V15W, V15Y, Y17C, Y17F, Y17G, Y17H, Y171, Y17K, Y17L, Y17M, Y17N, Y17Q, Y17R, Y17S, Y17T, Y17V, Y17W, R19D, R19H, R19K, R19M, R19T, R19Y, N21A, N21C, N21F, N21G, N21H, N21I, N21K, N21L, N21M, N21P, N21Q, N21R, N21S, N21T, N21W, and N21Y;

preferably

W4A, Y5H, Y5N, Y5R, V6A, V6F, V8A, Y9K, Y9R, G12R, G12K, V13A, V151, V15S, and Y17H.

In another aspect, the variant comprises the substitutions Y5N+Y17H of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y5N+Y9R of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y5N+Y9K of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y17H+Y9R of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y17H+Y9K of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y5N+Y17H+Y9R of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y5N+Y17H+Y9K of the mature peptide of SEQ ID NO: 2.

In another aspect, the variant comprises the substitutions Y5N+V6A+Y9K or V8A+Y9R+V13A or Y5N+Y9R+Y17H or Y9K+V155 or W4A+Y5R+Y9K or Y5N+G12R+Y17H or Y5N+V6F+Y17H or Y5N+V15I+Y17H or Y5H+V8A+Y9R or Y5N+G12K+Y17H of the mature peptide of SEQ ID NO: 2.

Essential amino acids in a parent can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for antimicrobial activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The identities of essential amino acids can also be inferred from analysis of identities with peptides that are related to the parent.

Polynucleotides

The present invention also relates to isolated polynucleotides that encode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

A polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized by a host cell for expression of the polynucleotide. The promoter sequence contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular peptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are the promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter including a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include modified promoters including the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the variant-encoding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the variant. However, any signal peptide coding region that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that encodes a propeptide positioned at the N-terminus of a variant. The resultant peptide is known as a proenzyme or propeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to an active peptide by catalytic or autocatalytic cleavage of the propeptide from the propeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide regions are present at the N-terminus of a variant, the propeptide region is positioned next to the N-terminus of the variant and the signal peptide region is positioned next to the N-terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra) to obtain substantially pure variants.

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminurn, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the peptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that are specific for the variants. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant.

The variant may be recovered by methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing a variant is used as a source of the variant.

In Vitro Synthesis

The polypeptides of the invention may also be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms) e.g. D-alanine and D-isoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g. reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like.

If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

Plants

The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising a polynucleotide of the present invention so as to express and produce the variant in recoverable quantities. The variant may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the variant may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats.

Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (several) expression constructs encoding a variant into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a variant operably linked with appropriate regulatory sequences required for expression of the polynucleotide in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying plant cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the variant is desired to be expressed. For instance, the expression of the gene encoding a variant may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higher expression of a variant in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the polynucleotide encoding a variant. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformation methods for use in accordance with the present disclosure include those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype with a construct prepared according to the present invention, transgenic plants may be made by crossing a plant having the construct to a second plant lacking the construct. For example, a construct encoding a variant can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present invention encompasses not only a plant directly regenerated from cells which have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a DNA construct prepared in accordance with the present invention, or a portion of a DNA construct prepared in accordance with the present invention. Crossing results in the introduction of a transgene into a plant line by cross pollinating a starting line with a donor plant line. Non-limiting examples of such steps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

The present invention also relates to methods of producing a variant of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and (b) recovering the variant.

Methods and Uses

The present invention is also directed to methods for using the polypeptides having antimicrobial activity. The antimicrobial polypeptides are typically useful at any locus subject to contamination by microorganisms. Typically, loci are in aqueous systems such as cooling water systems, where microorganisms need to be killed or where their growth needs to be controlled. However, the present invention may also be used in all applications for which known antimicrobial compositions are useful, such as protection of wood, latex, adhesive, glue, paper, cardboard, textile, leather and feed.

Other uses include preservation of foods, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products or food ingredients.

In general it is contemplated that the antimicrobial polypeptides of the present invention are useful for cleaning, disinfecting or inhibiting microbial growth on any surface. Examples of surfaces, which may advantageously be contacted with the antimicrobial polypeptides of the invention, are surfaces of process equipment used e.g. dairies, chemical or pharmaceutical process plants. The antimicrobial polypeptides of the invention should be used in an amount, which is effective for cleaning, disinfecting or inhibiting microbial growth on the surface in question.

The antimicrobial polypeptides of the invention may additionally be used for cleaning surfaces and cooking utensils in food processing plants and in any area in which food is prepared or served such as hospitals, nursing homes and restaurants.

The invention also relates to the use of an antimicrobial polypeptide or composition of the invention as a medicament. Further, an antimicrobial polypeptide or composition of the invention may also be used for the manufacture of a medicament for controlling or combating microorganisms, such as fungal organisms or bacteria, preferably gram negative bacteria.

The composition and antimicrobial polypeptide of the invention may be used as an antimicrobial veterinarian or human therapeutic or prophylactic agent. Thus, the composition and antimicrobial polypeptide of the invention may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of microbial infections, such as bacterial or fungal infections, preferably gram positive bacterial infections. In particular the microbial infections may be associated with lung diseases including, but not limited to, tuberculosis, pneumonia and cystic fibrosis; skin infections and infections in the eye or the mouth; and sexually transmitted diseases including, but not limited to, gonorrhea and chlamydia.

The invention also relates to wound healing compositions or products such as bandages, medical devices such as, e.g., catheters.

The composition of the invention comprises an effective amount of the antimicrobial polypeptide of the invention.

The term “effective amount” when used herein is intended to mean an amount of the antimicrobial polypeptides of the invention, which is sufficient to inhibit growth of the microorganisms in question.

Formulations of the antimicrobial polypeptides of the invention are administered to a host suffering from or predisposed to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism, preferably it will be localized. Generally the dose of the antimicrobial polypeptides of the invention will be sufficient to decrease the microbial population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs of killing. The compounds of the present invention are administered at a dosage that reduces the microbial population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use. The antimicrobial polypeptides of the invention are particularly useful for killing gram negative bacteria, including Pseudomonas aeruginosa, and Chlamydia trachomatis; and gram-positive bacteria, including streptococci such as Streptococcus pneumonia, S. uberis, S. hyointestinalis, S. pyogenes and S. agalactiae; and staphylococci such as Staphylococcus aureus, S. epidermidis, S. simulans, S. xylosus and S. carnosus.

Formulations of the antimicrobial polypeptides of the invention may be administered to a host suffering from or predisposed to a microbial lung infection, such as pneumonia; or to a microbial wound infection, such as a bacterial wound infection.

Formulations of the antimicrobial polypeptides of the invention may also be administered to a host suffering from or predisposed to a skin infection, such as acne, atopic dermatitis or seborrheic dermatitis; preferably the skin infection is a bacterial skin infection, e.g. caused by Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes, Pityrosporum ovale or Malassezia furfur.

The antimicrobial polypeptides of the invention are also useful for in vitro formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, the antimicrobial polypeptides of the invention may be added to animal and/or human food preparations; or they may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture.

The susceptibility of a particular microbe to killing with the antimicrobial polypeptides of the invention may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with the antimicrobial polypeptide at varying concentrations for a period of time sufficient to allow the protein to act, usually between about one hour and one day. The viable microbes are then counted, and the level of killing determined.

Microbes of interest include, but are not limited to, Gram-negative bacteria, for example: Citrobacter sp.; Enterobacter sp.; Escherichia sp., e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium; Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Y. enterocolitica; Franciscella sp.; Pasturella sp.; Vibrio sp., e.g. V. cholerae, V. parahemolyticus; Campylobacter sp., e.g. C. jejuni; Haemophilus sp., e.g. H. influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other bacteria of interest include Legionella sp., e.g. L. pneumophila; Listeria sp., e.g. L. monocytogenes; Mycoplasma sp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M. leprae; Treponema sp., e.g. T. pallidum; Borrelia sp., e.g. B. burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., e.g. H. pylori, etc.

Non-bacterial pathogens of interest include fungal and protozoan pathogens, e.g. Plasmodia sp., e.g. P. falciparum, Trypanosoma sp., e.g. T. brucei; shistosomes; Entaemoeba sp., Cryptococcus sp., Candida sp., e.g. C. albicans; etc.

Various methods for administration may be employed. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific antimicrobial polypeptide to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration. For example, the carboxy terminus may be amidated.

Formulations

The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The antimicrobial polypeptides of the invention may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.

The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.). In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing the antimicrobial polypeptides of the invention is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 pg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will be normally be neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

For preparing the liposomes, the procedure described by Kato et al., 1991, J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing peptides are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 seconds, the tube is placed in a warm water bath, from about 25-40° C. and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 seconds and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules.

Formulations with Other Active Agents

For use in the subject methods, the antimicrobial polypeptides of the invention may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with beta-lactamase inhibitors, cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; chloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.

Anti-mycotic agents are also useful, including polyenes, e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol and fluconazol. Antituberculotic drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in a formulation of the antimicrobial polypeptides of the invention, e.g. interferon gamma, tumor necrosis factor alpha, interleukin 12, etc.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

NZ17074 is the antimicrobial peptide of SEQ ID NO: 70.

Example 1 Isolation of Variants of SEQ ID NO: 2 Having Improved Antimicrobial Activity

The cDNA encoding SEQ ID NO: 2 was fused to the proregion of plectasin (see Mygind et al., 2005, Nature 437: 975-980) and the Mating Factor alpha-leader from Saccharomyces cerevisiae and introduced into the inducible S. cerevisiae expression vector, pYES2, and transformed into S. cerevisiae. This system takes advantage of the GAL1 promoter which is repressed by glucose and activated by galactose.

Several strategies were used for variant generation of the polynucleotide of SEQ ID NO:1. The resulting libraries were cloned and expressed in S. cerevisiae. Transformed clones were screened on a plate assay containing growth media supplemented with 1.5% galactose and 0.5% glucose and either horse blood (2.5-5%) or serum (5%), overlayed with the target organism, E. coli ATCC 10536 (See Raventos et al., 2005, Comb Chem High Throughput Screen. 8:219-33).

The plate assay conditions fully inhibited the activity of the antimicrobial peptide of SEQ ID NO: 2 (the parent antimicrobial peptide). Variants exhibiting improved antimicrobial activity (giving rise to clearing zones) in the presence of 2.5% blood, 5% blood or 5% serum were picked and sequenced, and are shown in Table 1.

Plate Assay Screening Procedure

Approximately, 300 Saccharomyces cerevisiae colonies expressing arenicin variants were spread on screening plates containing either horse blood (2.5% or 5%) or 5% horse serum (see composition of the plates below). Plates were incubated 3 hours at 30° C. to allow them to dry. Next, 25 ml overlay temperate at 42° C. was added to the plates. After the media had solidified, the plates were incubated 3 days at 30° C.

On day 4, plates were overlayed with pre-warmed media at 42° C. containing either 2.5% or 5% horse blood or 5% horse serum and the target bacteria, E. coli ATCC 10536 (see below for details on media composition). After the overlay solidified, plates were incubated 16 hours at 37° C. Next day, plates were colored by adding 10 ml of 1.5 mM MTT to the plates and incubated at room temperature for 30 minutes. Clones giving rise to clearing zones were picked and sequenced.

Plate and Media Composition

Three different types of screening plates a), b) and c) were used in the screening:

a) Plates+2.5% Horse Blood

The bottom layer contains 50 ml of 1.5% agarose+¼ SC media+2.5% blood+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+¼ SC media+2.5% blood+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+2.5% horse blood and 1.25×10⁶ colony forming units (cfu) of E. coli ATCC 10536.

B) Plates+5% Horse Blood

The bottom layer contains 50 ml of 1.5% agarose+¼ SC media+5% blood+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+¼ SC media+5% blood+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+5% horse blood and 1.25×10⁶ colony forming units (cfu) of E. coli ATCC 10536.

C) Plates+5% Horse Serum

The bottom layer contains 50 ml of 1.5% agarose+½ SC media+5% serum+1.5% galactose+0.5% glucose. The first overlay contains 25 ml of 1% agarose+½ SC media+5% serum+1.5% galactose+0.5% glucose. The top overlay contains 25 ml 0.2% MHB (#212322; BD)+1% agarose (Sigma A-4718)+5% horse serum and 1.25×10⁶ colony forming units (cfu) of E. coli ATCC 10536.

Composition of SC Media (450 ml)

Yeast Nitrogen Base w/o amino acids: 3.75 g Succinic acid: 5.65 g Sodium hydroxide: 3.4 g Casamino acid vitamin assay: 2.8 g

L-Tryptophan: 0.05 g Water: 450 ml

pH was adjusted to 6 and the media was autoclaved and diluted ¼ when preparing the blood plates and ½ when preparing the serum plates. MIT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma 13, 503-8)

Determination of the Protein Binding

Protein binding assays were performed as follows. The purified peptides were mixed with 90% serum and centrifuged through a 30 kDa filter. The ultra-filtrate and the non filtrated serum samples were quantified by HPLC measurements and the protein binding was subsequently calculated.

The antimicrobial peptide of SEQ ID NO: 2 (the parent antimicrobial peptide) exhibited a protein binding of 99.5% in this assay. As shown in Table 1, all exemplified variants exhibit a lower protein binding than the antimicrobial peptide of SEQ ID NO: 2.

TABLE 1 Variants exhibiting improved antimicrobial activity and reduced protein binding. SEQ ID Protein binding NO: Substitution Amino acid sequence (%) 3 F2A GACWYVCVYRNGVRVCYRRCN 86 4 W4A GFCAYVCVYRNGVRVCYRRCN 89 5 W4E GFCEYVCVYRNGVRVCYRRCN — 6 W4G GFCGYVCVYRNGVRVCYRRCN — 7 W4S GFCSYVCVYRNGVRVCYRRCN 68 8 W4T GFCAYVCVYRNGVRVCYRRCN — 9 W4Y GFCYYVCVYRNGVRVCYRRCN — 10 Y5K GFCWKVCVYRNGVRVCYRRCN 64 11 Y5N GFCWNVCVYRNGVRVCYRRCN 73 12 Y5R GFCWRVCVYRNGVRVCYRRCN — 13 V6A GFCWYACVYRNGVRVCYRRCN — 14 V6E GFCWYECVYRNGVRVCYRRCN — 15 V6G GFCWYGCVYRNGVRVCYRRCN 96 16 V6L GFCWYLCVYRNGVRVCYRRCN — 17 V6N GFCWYNCVYRNGVRVCYRRCN — 18 V6R GFCWYRCVYRNGVRVCYRRCN — 19 V6S GFCWYSCVYRNGVRVCYRRCN 89 20 V6W GFCWYWCVYRNGVRVCYRRCN 99 21 V8A GFCWYVCAYRNGVRVCYRRCN   98.5 22 V8G GFCWYVCGYRNGVRVCYRRCN — 23 V8H GFCWYVCHYRNGVRVCYRRCN 98 24 V8S GFCWYVCSYRNGVRVCYRRCN 85 25 V8Y GFCWYVCYYRNGVRVCYRRCN 98 26 Y9I GFCWYVCVIRNGVRVCYRRCN — 27 Y9K GFCWYVCVKRNGVRVCYRRCN — 28 Y9R GFCWYVCVRRNGVRVCYRRCN 92 29 V13A GFCWYVCVYRNGARVCYRRCN 99 30 V13G GFCWYVCVYRNGGRVCYRRCN 99 31 V13K GFCWYVCVYRNGKRVCYRRCN — 32 V13L GFCWYVCVYRNGLRVCYRRCN — 33 V13P GFCWYVCVYRNGPRVCYRRCN 99 34 V13Q GFCWYVCVYRNGQRVCYRRCN — 36 V13R GFCWYVCVYRNGRRVCYRRCN — 36 V13S GFCWYVCVYRNGSRVCYRRCN 60 37 V15A GFCWYVCVYRNGVRACYRRCN — 38 V15G GFCWYVCVYRNGVRGCYRRCN 93 39 V15H GFCWYVCVYRNGVRHCYRRCN 98 40 V15K GFCWYVCVYRNGVRKCYRRCN — 41 V15N GFCWYVCVYRNGVRNCYRRCN 97 42 V15Q GFCWYVCVYRNGVRQCYRRCN 96 43 V15R GFCWYVCVYRNGVRRCYRRCN — 44 V15S GFCWYVCVYRNGVRSCYRRCN 85 45 V15T GFCWYVCVYRNGVRTCYRRCN 98 46 Yl7H GFCWYVCVYRNGVRVCHRRCN 88 47 Yl7K GFCWYVCVYRNGVRVCKRRCN 89 48 Yl7N GFCWYVCVYRNGVRVCNRRCN 68 49 Yl7R GFCWYVCVYRNGVRVCRRRCN — 50 N21H GFCWYVCVYRNGVRVCYRRCH — 51 N21K GFCWYVCVYRNGVRVCYRRCK — 52 N21R GFCWYVCVYRNGVRVCYRRCR — 53 N21S GFCWYVCVYRNGVRVCYRRCS 99 54 N21T GFCWYVCVYRNGVRVCYRRCT 99 55 G1R + N21R RFCWYVCVYRNGVRVCYRRCR — 56 W4F + Y17R GFCFYVCVYRNGVRVCRRRCN 94 57 W4A + Y5H GFCAHVCVYRNGVRVCYRRCN — 58 W4A + Y5R GFCARVCVYRNGVRVCYRRCN — 59 W4F + Y9R GFCFYVCVRRNGVRVCYRRCN — 60 W4G + Y5H GFCGHVCVYRNGVRVCYRRCN 64 61 W4G + Y5R GFCGRVCVYRNGVRVCYRRCN 72 62 W4S + V15A GFCSYVCVYRNGVRACYRRCN — 63 W4S + Y5R GFCSRVCVYRNGVRVCYRRCN — 64 W4Y + Y5R GFCYRVCVYRNGVRVCYRRCN — 65 Y5F + V15Q GFCWFVCVYRNGVRQCYRRCN — 66 Y5H + V15S GFCWHVCVYRNGVRSCYRRCN — 67 Y5H + Y17R GFCWHVCVYRNGVRVCRRRCN — 68 Y5K + Y17S GFCWKVCVYRNGVRVCSRRCN 87 69 Y5N + V15S GFCWNVCVYRNGVRSCYRRCN — 70 Y5N + Y17H GFCWNVCVYRNGVRVCHRRCN 79 71 Y5R + V15P GFCWRVCVYRNGVRPCYRRCN — 72 Y5R + V6A GFCWRACVYRNGVRVCYRRCN 97 73 Y5R + V8G GFCWRVCGYRNGVRVCYRRCN — 74 Y5R + V8H GFCWRVCHYRNGVRVCYRRCN 99 75 Y5R + V8S GFCWRVCSYRNGVRVCYRRCN — 76 Y5R + Y17H GFCWRVCVYRNGVRVCHRRCN 92 77 Y5R + Y17N GFCWRVCVYRNGVRVCNRRCN — 78 Y5S + V15S GFCWSVCVYRNGVRSCYRRCN — 79 V6A + V13A GFCWYACVYRNGARVCYRRCN — 80 V6A + V13K GFCWYACVYRNGKRVCYRRCN 96 81 V6A + V15A GFCWYACVYRNGVRACYRRCN — 82 V6A + Y17H GFCWYACVYRNGVRVCHRRCN — 83 V6M + V8G GFCWYMCGYRNGVRVCYRRCN — 84 V8A + V15A GFCWYVCAYRNGVRACYRRCN — 85 V8S + V13K GFCWYVCSYRNGKRVCYRRCN — 86 Y9K + V13A GFCWYVCVKRNGARVCYRRCN — 87 Y9K + V15S GFCWYVCVKRNGVRSCYRRCN 93 88 R10K + V15S GFCWYVCVYKNGVRSCYRRCN — 89 R10K + Y17H GFCWYVCVYKNGVRVCHRRCN 90 90 R10P + V13G GFCWYVCVYPNGGRVCYRRCN — 91 N11R + V15Q GFCWYVCVYRRGVRQCYRRCN — 92 V13A + Y17C GFCWYVCVYRNGARVCCRRCN — 93 V13G + V15K GFCWYVCVYRNGGRKCYRRCN — 94 V15L + Y17H GFCWYVCVYRNGVRLCHRRCN — 95 G1A + Y5N + Y17H AFCWNVCVYRNGVRVCHRRCN — 96 G1D + Y5N + Y17H DFCWNVCVYRNGVRVCHRRCN — 97 G1F + Y5N + Y17H FFCWNVCVYRNGVRVCHRRCN — 98 G1H + Y5N + Y17H HFCWNVCVYRNGVRVCHRRCN 77 99 G1I + Y5N + Y17H IFCWNVCVYRNGVRVCHRRCN — 100 G1K + Y5N + Y17H KFCWNVCVYRNGVRVCHRRCN 78 101 G1M + Y5N + Y17H MFCWNVCVYRNGVRVCHRRCN 81 102 G1Q + Y5N + Y17H QFCWNVCVYRNGVRVCHRRCN — 103 G1R + Y5N + Y17H RFCWNVCVYRNGVRVCHRRCN — 104 G1S + Y5N + Y17H SFCWNVCVYRNGVRVCHRRCN — 105 G1T + Y5N + Y17H TFCWNVCVYRNGVRVCHRRCN — 106 G1V + Y5N + Y17H VFCWNVCVYRNGVRVCHRRCN — 107 G1W + Y5N + Y17H WFCWNVCVYRNGVRVCHRRCN — 108 GlY + Y5N + Y17H YFCWNVCVYRNGVRVCHRRCN — 109 F2G + Y5N + Y17H GGCWNVCVYRNGVRVCHRRCN — 110 F2H + Y5N + Y17H GHCWNVCVYRNGVRVCHRRCN — 111 F2I + Y5N + Y17H GICWNVCVYRNGVRVCHRRCN — 112 F2L + Y5N + Y17H GLCWNVCVYRNGVRVCHRRCN — 113 F2M + Y5N + Y17H GMCWNVCVYRNGVRVCHRRCN — 114 F2P + Y5N + Y17H GPCWNVCVYRNGVRVCHRRCN — 115 F2V + Y5N + Y17H GVCWNVCVYRNGVRVCHRRCN — 116 F2W + Y5N + Y17H GWCWNVCVYRNGVRVCHRRCN — 117 F2Y + Y5N + Y17H GYCWNVCVYRNGVRVCHRRCN — 118 C3L + W4Q + Y17H GFLQYVCVYRNGVRVCHRRCN — 119 W4A + V6A + Y9K GFCAYACVKRNGVRVCYRRCN — 120 W4A + Y5K + V15S GFCAKVCVYRNGVRSCYRRCN 69 121 W4A + Y5R + V15A GFCARVCVYRNGVRACYRRCN — 122 W4A + Y5R + V15S GFCARVCVYRNGVRSCYRRCN 81 123 W4A + Y5R + V15T GFCARVCVYRNGVRTCYRRCN — 124 W4A + Y5R + V8S GFCARVCSYRNGVRVCYRRCN — 125 W4A + Y5R + Y9K GFCARVCVKRNGVRVCYRRCN 90 126 W4A + Y5W + V15Q GFCAWVCVYRNGVRQCYRRCN — 127 W4A + Y9R + V15S GFCAYVCVRRNGVRSCYRRCN — 128 W4F + V8A + V15S GFCFYVCAYRNGVRSCYRRCN — 129 W4F + Y5H + V15S GFCFHVCVYRNGVRSCYRRCN — 130 W4F + Y5N + V15S GFCFNVCVYRNGVRSCYRRCN — 131 W4F + Y5N + Y17H GFCFNVCVYRNGVRVCHRRCN — 132 W4F + Y5R + V15K GFCFRVCVYRNGVRKCYRRCN — 133 W4F + Y5R + V15Q GFCFRVCVYRNGVRQCYRRCN — 134 W4F + Y5R + V15S GFCFRVCVYRNGVRSCYRRCN — 135 W4F + Y5R + Y17H GFCFRVCVYRNGVRVCHRRCN — 136 W4F + Y5R + Y17Q GFCFRVCVYRNGVRVCQRRCN — 137 W4F + Y9K + Y17H GFCFYVCVKRNGVRVCHRRCN 86 138 W4G + Y5H + N21A GFCGHVCVYRNGVRVCYRRCA — 139 W4G + Y5H + N21K GFCGHVCVYRNGVRVCYRRCK — 140 W4G + Y5H + N21L GFCGHVCVYRNGVRVCYRRCL — 141 W4G + Y5H + N21M GFCGHVCVYRNGVRVCYRRCM — 142 W4G + Y5H + N21P GFCGHVCVYRNGVRVCYRRCP — 143 W4G + Y5H + N21R GFCGHVCVYRNGVRVCYRRCR — 144 W4G + Y5H + N21S GFCGHVCVYRNGVRVCYRRCS 68 145 W4G + Y5H + N21Y GFCGHVCVYRNGVRVCYRRCY — 146 W4G + Y5H + V15A GFCGHVCVYRNGVRACYRRCN — 147 W4G + Y5H + V15F GFCGHVCVYRNGVRFCYRRCN — 148 W4G + Y5H + V15G GFCGHVCVYRNGVRGCYRRCN — 149 W4G + Y5H + V15H GFCGHVCVYRNGVRHCYRRCN — 150 W4G + Y5H + V15I GFCGHVCVYRNGVRICYRRCN — 151 W4G + Y5H + V15L GFCGHVCVYRNGVRLCYRRCN — 152 W4G + Y5H + V15M GFCGHVCVYRNGVRMCYRRCN — 153 W4G + Y5H + V15N GFCGHVCVYRNGVRNCYRRCN — 154 W4G + Y5H + V15Q GFCGHVCVYRNGVRQCYRRCN — 155 W4G + Y5H + V15R GFCGHVCVYRNGVRRCYRRCN 83 156 W4G + Y5H + V15S GFCGHVCVYRNGVRSCYRRCN — 157 W4G + Y5H + V15T GFCGHVCVYRNGVRTCYRRCN — 158 W4G + Y5H + V15W GFCGHVCVYRNGVRWCYRRCN — 159 W4G + Y5H + V15Y GFCGHVCVYRNGVRYCYRRCN — 160 W4G + Y5H + Y17F GFCGHVCVYRNGVRVCFRRCN — 161 W4G + Y5H + Y17G GFCGHVCVYRNGVRVCGRRCN — 162 W4G + Y5H + Y17I GFCGHVCVYRNGVRVCIRRCN — 163 W4G + Y5H + Y17L GFCGHVCVYRNGVRVCLRRCN — 164 W4G + Y5H + Y17M GFCGHVCVYRNGVRVCMRRCN — 165 W4G + Y5H + Y17T GFCGHVCVYRNGVRVCTRRCN — 166 W4G + Y5H + Y17V GFCGHVCVYRNGVRVCVRRCN — 167 W4G + Y5H + Y17W GFCGHVCVYRNGVRVCWRRCN — 168 W4G + Y5K + V15H GFCGKVCVYRNGVRHCYRRCN — 169 W4G + Y5R + V15L GFCGRVCVYRNGVRLCYRRCN — 170 W4G + Y5R + V15R GFCGRVCVYRNGVRRCYRRCN 87 171 W4G + Y5R + V15T GFCGRVCVYRNGVRTCYRRCN 84 172 W4G + Y5R + Y9R GFCGRVCVRRNGVRVCYRRCN 88 173 W4G + Y5S + V15A GFCGSVCVYRNGVRACYRRCN — 174 W4G + Y5S + V15K GFCGSVCVYRNGVRKCYRRCN — 175 W4G + Y5S + V15R GFCGSVCVYRNGVRRCYRRCN — 176 W4G + Y9R + V15S GFCGYVCVRRNGVRSCYRRCN — 177 W4I + Y5N + Y17H GFCINVCVYRNGVRVCHRRCN — 178 W4L + Y5H + V15Q GFCLHVCVYRNGVRQCYRRCN — 179 W4L + Y5K + V15K GFCLKVCVYRNGVRKCYRRCN — 180 W4L + Y5R + V15Q GFCLRVCVYRNGVRQCYRRCN — 181 W4L + Y5R + V15S GFCLRVCVYRNGVRSCYRRCN — 182 W4M + Y5E + Y17T GFCMEVCVYRNGVRVCTRRCN — 183 W4M + Y5H + V15S GFCMHVCVYRNGVRSCYRRCN — 184 W4M + Y5N + Y17H GFCMNVCVYRNGVRVCHRRCN — 185 W4M + Y5R + V15T GFCMRVCVYRNGVRTCYRRCN — 186 W4M + Y5S + V15Q GFCMSVCVYRNGVRQCYRRCN — 187 W4M + Y5S + V15R GFCMSVCVYRNGVRRCYRRCN — 188 W4N + Y5R + V15I GFCNRVCVYRNGVRICYRRCN — 189 W4S + Y5K + V15R GFCSKVCVYRNGVRRCYRRCN — 190 W4S + Y5R + V15I GFCSRVCVYRNGVRICYRRCN — 191 W4T + Y5N + V15A GFCTNVCVYRNGVRACYRRCN — 192 W4T + Y5R + V15R GFCTRVCVYRNGVRRCYRRCN — 193 W4T + Y5R + V151 GFCTRVCVYRNGVRTCYRRCN — 194 W4T + Y5S + V15H GFCTSVCVYRNGVRHCYRRCN — 195 W4T + Y9K + V15K GFCTYVCVKRNGVRKCYRRCN 95 196 W4V + Y5H + V15P GFCVHVCVYRNGVRPCYRRCN — 197 W4V + Y5H + Y17H GFCVHVCVYRNGVRVCHRRCN — 198 W4V + Y5K + V15Q GFCVKVCVYRNGVRQCYRRCN — 199 W4V + Y5N + Y17H GFCVNVCVYRNGVRVCHRRCN — 200 W4V + Y5R + V15G GFCVRVCVYRNGVRGCYRRCN — 201 W4V + Y5R + V15P GFCVRVCVYRNGVRPCYRRCN — 202 W4V + Y5R + V15Q GFCVRVCVYRNGVRQCYRRCN — 203 W4V + Y5R + V15R GFCVRVCVYRNGVRRCYRRCN — 204 W4V + Y5R + V15T GFCVRVCVYRNGVRTCYRRCN — 205 W4Y + Y5H + V15Y GFCYHVCVYRNGVRYCYRRCN — 206 W4Y + Y5K + V15S GFCYKVCVYRNGVRSCYRRCN 86 207 W4Y + Y5N + V15R GFCYNVCVYRNGVRRCYRRCN 91 208 W4Y + Y5N + Y17H GFCYNVCVYRNGVRVCHRRCN — 209 W4Y + Y5R + V15T GFCYRVCVYRNGVRTCYRRCN 89 210 W4Y + Y5R + Y17H GFCYRVCVYRNGVRVCHRRCN 89 211 W4Y + Y5R + Y17S GFCYRVCVYRNGVRVCSRRCN 96 212 W4Y + Y5W + V15S GFCYWVCVYRNGVRSCYRRCN — 213 Y5H + V13A + V15S GFCWHVCVYRNGARSCYRRCN 95 214 Y5H + V13S + V15S GFCWHVCVYRNGSRSCYRRCN — 215 Y5H + V15S + Y17S GFCWHVCVYRNGVRSCSRRCN — 216 Y5H + V8A + V13K GFCWHVCAYRNGKRVCYRRCN — 217 Y5H + V8A + V15S GFCWHVCAYRNGVRSCYRRCN — 218 Y5H + V8A + Y9R GFCWHVCARRNGVRVCYRRCN 99 219 Y5H + V8H + G12S GFCWHVCHYRNSVRVCYRRCN 99 220 Y5H + Y9S + V15K GFCWHVCVSRNGVRKCYRRCN — 221 Y5K + Y9S + Y17S GFCWKVCVSRNGVRVCSRRCN — 222 Y5N + G12A + Y17H GFCWNVCVYRNAVRVCHRRCN — 223 Y5N + Gl2D + Y17H GFCWNVCVYRNDVRVCHRRCN — 224 Y5N + G12E + Y17H GFCWNVCVYRNEVRVCHRRCN — 225 Y5N + Gl2F + Y17H GFCWNVCVYRNFVRVCHRRCN — 226 Y5N + Gl2H + Y17H GFCWNVCVYRNHVRVCHRRCN — 227 Y5N + G12K + Y17H GFCWNVCVYRNKVRVCHRRCN 80 228 Y5N + Gl2R + Y17H GFCWNVCVYRNRVRVCHRRCN 86 229 Y5N + G12Y + Y17H GFCWNVCVYRNYVRVCHRRCN 91 230 Y5N + N11A + Y17H GFCWNVCVYRAGVRVCHRRCN 71 231 Y5N + Nl1G + Y17H GFCWNVCVYRGGVRVCHRRCN 66 232 Y5N + Nl1H + Y17H GFCWNVCVYRHGVRVCHRRCN 85 233 Y5N + N11Q + Y17H GFCWNVCVYRQGVRVCHRRCN 78 234 Y5N + Nl1R + Y17H GFCWNVCVYRRGVRVCHRRCN 86 235 Y5N + V13A + Y17H GFCWNVCVYRNGARVCHRRCN 64 236 Y5N + V13F + Y17H GFCWNVCVYRNGFRVCHRRCN — 237 Y5N + V13H + Y17H GFCWNVCVYRNGHRVCHRRCN 40 238 Y5N + V13Q + Y17H GFCWNVCVYRNGQRVCHRRCN — 239 Y5N + V13R + Y17H GFCWNVCVYRNGRRVCHRRCN 65 240 Y5N + V13T + Y17H GFCWNVCVYRNGTRVCHRRCN — 241 Y5N + V13W + Y17H GFCWNVCVYRNGWRVCHRRCN — 242 Y5N + V13Y + Y17H GFCWNVCVYRNGYRVCHRRCN 83 243 Y5N + V15A + Y17H GFCWNVCVYRNGVRACHRRCN — 244 Y5N + V15C + Y17H GFCWNVCVYRNGVRCCHRRCN — 245 Y5N + V15F + Y17H GFCWNVCVYRNGVRFCHRRCN 85 246 Y5N + V15G + Y17H GFCWNVCVYRNGVRGCHRRCN 79 247 Y5N + V15H + Y17H GFCWNVCVYRNGVRHCHRRCN — 248 Y5N + V15I + Y17H GFCWNVCVYRNGVRICHRRCN 58 249 Y5N + V15L + Y17H GFCWNVCVYRNGVRLCHRRCN 83 250 Y5N + V15M + Y17H GFCWNVCVYRNGVRMCHRRCN — 251 Y5N + V15N + Y17H GFCWNVCVYRNGVRNCHRRCN 71 252 Y5N + V15R + Y17H GFCWNVCVYRNGVRRCHRRCN 76 253 Y5N + V15W + Y17H GFCWNVCVYRNGVRWCHRRCN 94 254 Y5N + V15Y + Y17H GFCWNVCVYRNGVRYCHRRCN 77 255 Y5N + V6A + V15N GFCWNACVYRNGVRNCYRRCN — 256 Y5N + V6A + V15S GFCWNACVYRNGVRSCYRRCN — 257 Y5N + V6A + Y17H GFCWNACVYRNGVRVCHRRCN — 258 Y5N + V6A + Y9K GFCWNACVKRNGVRVCYRRCN 89 259 Y5N + V6C + Y17H GFCWNCCVYRNGVRVCHRRCN — 260 Y5N + V6F + Y17H GFCWNFCVYRNGVRVCHRRCN 84 261 Y5N + V6H + Y17H GFCWNHCVYRNGVRVCHRRCN — 262 Y5N + V61 + Y17H GFCWNICVYRNGVRVCHRRCN 88 263 Y5N + V6L + Y17H GFCWNLCVYRNGVRVCHRRCN 80 264 Y5N + V6M + Y17H GFCWNMCVYRNGVRVCHRRCN — 265 Y5N + V6Q + Y17H GFCWNQCVYRNGVRVCHRRCN — 266 Y5N + V6T + Y17H GFCWNTCVYRNGVRVCHRRCN — 267 Y5N + V6W + Y17H GFCWNWCVYRNGVRVCHRRCN 85 268 Y5N + V6Y + Y17H GFCWNYCVYRNGVRVCHRRCN — 269 Y5N + V8A + V13A GFCWNVCAYRNGARVCYRRCN — 270 Y5N + V8A + V15S GFCWNVCAYRNGVRSCYRRCN — 271 Y5N + V8A + Y17H GFCWNVCAYRNGVRVCHRRCN — 272 Y5N + V8F + Y17H GFCWNVCFYRNGVRVCHRRCN 74 273 Y5N + V8G + Y17H GFCWNVCGYRNGVRVCHRRCN — 274 Y5N + V81 + Y17H GFCWNVCIYRNGVRVCHRRCN — 275 Y5N + V8L + Y17H GFCWNVCLYRNGVRVCHRRCN 80 276 Y5N + V8W + Y17H GFCWNVCWYRNGVRVCHRRCN — 277 Y5N + V8Y + Y17H GFCWNVCYYRNGVRVCHRRCN — 278 Y5N + Y17H + N2lA GFCWNVCVYRNGVRVCHRRCA — 279 Y5N + Y17H + N21C GFCWNVCVYRNGVRVCHRRCC — 280 Y5N + Y17H + N21F GFCWNVCVYRNGVRVCHRRCF — 281 Y5N + Y17H + N21G GFCWNVCVYRNGVRVCHRRCG 66 282 Y5N + Y17H + N21H GFCWNVCVYRNGVRVCHRRCH 82 283 Y5N + Y17H + N21I GFCWNVCVYRNGVRVCHRRCI — 284 Y5N + Y17H + N21K GFCWNVCVYRNGVRVCHRRCK 72 285 Y5N + Y17H + N21L GFCWNVCVYRNGVRVCHRRCL — 286 Y5N + Y17H + N21M GFCWNVCVYRNGVRVCHRRCM — 287 Y5N + Y17H + N21P GFCWNVCVYRNGVRVCHRRCP — 288 Y5N + Y17H + N21Q GFCWNVCVYRNGVRVCHRRCQ — 289 Y5N + Y17H + N21R GFCWNVCVYRNGVRVCHRRCR 83 290 Y5N + Y17H + N21S GFCWNVCVYRNGVRVCHRRCS — 291 Y5N + Y17H + N21W GFCWNVCVYRNGVRVCHRRCW 99 292 Y5N + Y17H + N21Y GFCWNVCVYRNGVRVCHRRCY — 293 Y5N + Y17H + R19D GFCWNVCVYRNGVRVCHRDCN — 294 Y5N + Y17H + R19H GFCWNVCVYRNGVRVCHRHCN — 295 Y5N + Y17H + R19K GFCWNVCVYRNGVRVCHRKCN — 296 Y5N + Y17H + R19M GFCWNVCVYRNGVRVCHRMCN — 297 Y5N + Y17H + R191 GFCWNVCVYRNGVRVCHRTCN — 298 Y5N + Y17H + R19Y GFCWNVCVYRNGVRVCHRYCN — 299 Y5N + Y9A + Y17H GFCWNVCVARNGVRVCHRRCN 74 300 Y5N + Y9D + Y17H GFCWNVCVDRNGVRVCHRRCN — 301 Y5N + Y9F + Y17H GFCWNVCVFRNGVRVCHRRCN — 302 Y5N + Y9G + Y17H GFCWNVCVGRNGVRVCHRRCN — 303 Y5N + Y9H + Y17H GFCWNVCVHRNGVRVCHRRCN 43 304 Y5N + Y9I + Y17H GFCWNVCVIRNGVRVCHRRCN — 305 Y5N + Y9K + Y17H GFCWNVCVKRNGVRVCHRRCN 76 306 Y5N + Y9M + Y17H GFCWNVCVMRNGVRVCHRRCN — 307 Y5N + Y9Q + Y17H GFCWNVCVQRNGVRVCHRRCN — 308 Y5N + Y9R + Y17H GFCWNVCVRRNGVRVCHRRCN 94 309 Y5N + Y9S + Y17H GFCWNVCVSRNGVRVCHRRCN — 310 Y5N + Y9I + Y17H GFCWNVCVTRNGVRVCHRRCN — 311 Y5N + Y9V + Y17H GFCWNVCVVRNGVRVCHRRCN — 312 Y5N + Y9W + Y17H GFCWNVCVWRNGVRVCHRRCN — 313 Y5R + V13A + V15K GFCWRVCVYRNGARKCYRRCN 98 314 Y5R + V13A + Y17S GFCWRVCVYRNGARVCSRRCN — 315 Y5R + V15K + Y17H GFCWRVCVYRNGVRKCHRRCN 93 316 Y5R + V15S + Y17H GFCWRVCVYRNGVRSCHRRCN 90 317 Y5R + V15S + Y17S GFCWRVCVYRNGVRSCSRRCN — 318 Y5R + V6A + G12S GFCWRACVYRNSVRVCYRRCN — 319 Y5R + V6A + V15A GFCWRACVYRNGVRACYRRCN 40 320 Y5R + V6A + V15S GFCWRACVYRNGVRSCYRRCN 88 321 Y5R + V6A + Y17H GFCWRACVYRNGVRVCHRRCN — 322 Y5R + V6A + Y9K GFCWRACVKRNGVRVCYRRCN — 323 Y5R + V6C + V15S GFCWRCCVYRNGVRSCYRRCN — 324 Y5R + V6S + Y17H GFCWRSCVYRNGVRVCHRRCN — 325 Y5R + V8A + V15S GFCWRVCAYRNGVRSCYRRCN — 326 Y5R + V8G + Y17H GFCWRVCGYRNGVRVCHRRCN — 327 Y5R + V8H + G12S GFCWRVCHYRNSVRVCYRRCN 98 328 Y5R + V8H + V13K GFCWRVCHYRNGKRVCYRRCN — 329 Y5R + V8S + V13A GFCWRVCSYRNGARVCYRRCN — 330 Y5R + V8S + V13S GFCWRVCSYRNGSRVCYRRCN — 331 Y5R + V8S + V15K GFCWRVCSYRNGVRKCYRRCN 95 332 Y5R + V8S + Y9R GFCWRVCSRRNGVRVCYRRCN — 333 Y5R + V8S + Y9S GFCWRVCSSRNGVRVCYRRCN — 334 Y5R + Y9R + V13A GFCWRVCVRRNGARVCYRRCN — 335 Y5R + Y9R + Y17H GFCWRVCVRRNGVRVCHRRCN 95 336 Y5R + Y9S + Y17H GFCWRVCVSRNGVRVCHRRCN — 337 Y5R + Y9S + Y17S GFCWRVCVSRNGVRVCSRRCN — 338 V6A + Gl2S + V13K GFCWYACVYRNSKRVCYRRCN — 339 V6A + V13A + V15S GFCWYACVYRNGARSCYRRCN — 340 V6A + V13K + V15A GFCWYACVYRNGKRACYRRCN 98 341 V6A + V13K + Y17H GFCWYACVYRNGKRVCHRRCN — 342 V6A + V15K + Y17H GFCWYACVYRNGVRKCHRRCN — 343 V6A + V8A + V15A GFCWYACAYRNGVRACYRRCN 74 344 V6R + V8H + R10S GFCWYRCHYSNGVRVCYRRCN — 345 V6S + Y9R + V15S GFCWYSCVRRNGVRSCYRRCN — 346 V6T + Y9K + V13L GFCWYTCVKRNGLRVCYRRCN 86 347 V8A + R10K + Yl7H GFCWYVCAYKNGVRVCHRRCN — 348 V8A + V15A + Y17H GFCWYVCAYRNGVRACHRRCN — 349 V8A + V15S + Y17H GFCWYVCAYRNGVRSCHRRCN — 350 V8A + Y9R + V13A GFCWYVCARRNGARVCYRRCN — 351 V8A + Y9R + V15S GFCWYVCARRNGVRSCYRRCN — 352 V8F + G12H + Y17H GFCWYVCFYRNHVRVCHRRCN — 353 Y9F + V15A + Y17H GFCWYVCVFRNGVRACHRRCN — 354 Y9K + V13A + Y17S GFCWYVCVKRNGARVCSRRCN — 355 Y9R + V15S + Y17S GFCWYVCVRRNGVRSCSRRCN — 356 R10K + V13K + V15S GFCWYVCVYKNGKRSCYRRCN — 357 V13K + V15A + Y17H GFCWYVCVYRNGKRACHRRCN — 358 G1R + Y5N + Nl1H + Y17H RFCWNVCVYRHGVRVCHRRCN — 359 G1R + Y5N + Y17H + R19H RFCWNVCVYRNGVRVCHRHCN 80 360 W4A + V6A + Y9K + V13A GFCAYACVKRNGARVCYRRCN — 361 W4A + Y5H + V13A + V15K GFCAHVCVYRNGARKCYRRCN — 362 W4A + Y5H + Y9K + V13A GFCAHVCVKRNGARVCYRRCN 76 363 W4A + Y5R + V8A + Y9K GFCARVCAKRNGVRVCYRRCN 80 364 W4A + Y5R + Y9K + V15S GFCARVCVKRNGVRSCYRRCN — 365 W4A + Y5R + Y9R + V15K GFCARVCVRRNGVRKCYRRCN — 366 W4A + Y5R + Y9R + V15S GFCARVCVRRNGVRSCYRRCN — 367 W4F + V6A + Y9R + V15S GFCFYACVRRNGVRSCYRRCN — 368 W4F + V8A + Y9S + V15S GFCFYVCASRNGVRSCYRRCN — 369 W4F + Y5H + V8A + Y17H GFCFHVCAYRNGVRVCHRRCN — 370 W4F + Y5H + V8S + V15K GFCFHVCSYRNGVRKCYRRCN — 371 W4F + Y5N + V6A + V15S GFCFNACVYRNGVRSCYRRCN — 372 W4F + Y5N + V8A + Y9R GFCFNVCARRNGVRVCYRRCN — 373 W4F + Y5R + V15K + Y17H GFCFRVCVYRNGVRKCHRRCN — 374 W4F + Y5R + V15S + Y17S GFCFRVCVYRNGVRSCSRRCN — 375 W4F + Y5R + V6A + Y9K GFCFRACVKRNGVRVCYRRCN — 376 W4F + Y5R + V6S + V13A GFCFRSCVYRNGARVCYRRCN — 377 W4F + Y5R + V6S + Y9R GFCFRSCVRRNGVRVCYRRCN 65 378 W4F + Y5R + V8A + V15K GFCFRVCAYRNGVRKCYRRCN — 379 W4F + Y5R + V8A + Y17H GFCFRVCAYRNGVRVCHRRCN — 380 W4F + Y5R + V8A + Y9S GFCFRVCASRNGVRVCYRRCN — 381 W4F + Y5R + Y9K + V15K GFCFRVCVKRNGVRKCYRRCN 98 382 W4F + Y9R + V15S + Y17H GFCFYVCVRRNGVRSCHRRCN — 383 W4G + Y5H + V15M + N21H GFCGHVCVYRNGVRMCYRRCH — 384 W4G + Y5K + Y9R + V13L GFCGKVCVRRNGLRVCYRRCN — 385 W4G + Y5R + V13K + V15A GFCGRVCVYRNGKRACYRRCN — 386 W4G + Y5R + V13K + V15A GFCGRVCVYRNGKRACYRRCN 81 387 W4G + Y5R + V13K + V15S GFCGRVCVYRNGKRSCYRRCN — 388 W4G + Y5R + Y9R + V15K GFCGRVCVRRNGVRKCYRRCN — 389 W4T + Y5R + Y9R + V13L GFCTRVCVRRNGLRVCYRRCN — 390 Y5H + R10K + V13K + V15S GFCWHVCVYKNGKRSCYRRCN 87 391 Y5H + V6A + Y9R + V15S GFCWHACVRRNGVRSCYRRCN — 392 Y5H + V8A + V13K + V15A GFCWHVCAYRNGKRACYRRCN — 393 Y5H + V8A + Y9K + V13L GFCWHVCAKRNGLRVCYRRCN — 394 Y5H + V8A + Y9R + V13A GFCWHVCARRNGARVCYRRCN 80 395 Y5H + V8A + Y9R + Y17H GFCWHVCARRNGVRVCHRRCN — 396 Y5H + V8S + Y9K + V13L GFCWHVCSKRNGLRVCYRRCN 92 397 Y5H + Y9R + V13A + V15K GFCWHVCVRRNGARKCYRRCN — 398 Y5H + Y9R + V13A + V15S GFCWHVCVRRNGARSCYRRCN — 399 Y5H + Y9R + V13A + Y17S GFCWHVCVRRNGARVCSRRCN — 400 Y5H + Y9S + V13L + V15K GFCWHVCVSRNGLRKCYRRCN — 401 Y5K + V6A + R10K + Y17H GFCWKACVYKNGVRVCHRRCN — 402 Y5K + V6A + V13A + V15S GFCWKACVYRNGARSCYRRCN — 403 Y5K + V6A + V8A + V13A GFCWKACAYRNGARVCYRRCN — 404 Y5K + Y9K + V13A + V15K GFCWKVCVKRNGARKCYRRCN — 405 Y5K + Y9R + V13A + Y17S GFCWKVCVRRNGARVCSRRCN — 406 Y5N + V6A + V8A + V13K GFCWNACAYRNGKRVCYRRCN — 407 Y5N + V6A + V8A + Y9R GFCWNACARRNGVRVCYRRCN 88 408 Y5N + V6A + Y9R + V15S GFCWNACVRRNGVRSCYRRCN — 409 Y5N + V6F + N11Y + Y17H GFCWNFCVYRYGVRVCHRRCN — 410 Y5N + V8A + N11Q + Y17H GFCWNVCAYRQGVRVCHRRCN — 411 Y5N + V8A + V13K + V15A GFCWNVCAYRNGKRACYRRCN 86 412 Y5N + Y9R + V13A + V15S GFCWNVCVRRNGARSCYRRCN — 413 Y5R + Gl2S + V13K + Y17H GFCWRVCVYRNSKRVCHRRCN — 414 Y5R + R10K + V13K + Y17H GFCWRVCVYKNGKRVCHRRCN — 415 Y5R + V6A + V13K + V15S GFCWRACVYRNGKRSCYRRCN — 416 Y5R + V6A + V15A + Y17H GFCWRACVYRNGVRACHRRCN — 417 Y5R + V6A + V8A + V13K GFCWRACAYRNGKRVCYRRCN 95 418 Y5R + V6A + V8A + V15A GFCWRACAYRNGVRACYRRCN 92 419 Y5R + V6A + V8A + Y17H GFCWRACAYRNGVRVCHRRCN — 420 Y5R + V6A + V8A + Y9R GFCWRACARRNGVRVCYRRCN — 421 Y5R + V6A + V8H + V15S GFCWRACHYRNGVRSCYRRCN — 422 Y5R + V6A + Y9R + V15K GFCWRACVRRNGVRKCYRRCN — 423 Y5R + V6S + V13A + V15S GFCWRSCVYRNGARSCYRRCN — 424 Y5R + V8A + Gl2S + V15S GFCWRVCAYRNSVRSCYRRCN — 425 Y5R + V8A + R10S + V13K GFCWRVCAYSNGKRVCYRRCN 69 426 Y5R + V8A + V13A + Yl7H GFCWRVCAYRNGARVCHRRCN — 427 Y5R + V8A + V13K + V15A GFCWRVCAYRNGKRACYRRCN — 428 Y5R + V8A + V13K + Yl7H GFCWRVCAYRNGKRVCHRRCN — 429 Y5R + V8A + V15A + Yl7H GFCWRVCAYRNGVRACHRRCN — 430 Y5R + V8A + V15S + Y17H GFCWRVCAYRNGVRSCHRRCN — 431 Y5R + V8A + Y9K + V15K GFCWRVCAKRNGVRKCYRRCN — 432 Y5R + V8A + Y9K + V15S GFCWRVCAKRNGVRSCYRRCN — 433 Y5R + V8A + Y9K + Y17H GFCWRVCAKRNGVRVCHRRCN — 434 Y5R + V8A + Y9R + V15S GFCWRVCARRNGVRSCYRRCN — 435 Y5R + V8A + Y9S + V13L GFCWRVCASRNGLRVCYRRCN 85 436 Y5R + V8G + Gl2S + V13K GFCWRVCGYRNSKRVCYRRCN — 437 Y5R + V8H + Gl2S + V13K GFCWRVCHYRNSKRVCYRRCN — 438 Y5R + V8H + R10K + V13K GFCWRVCHYKNGKRVCYRRCN — 439 Y5R + V8H + R10S + V13K GFCWRVCHYSNGKRVCYRRCN 92 440 Y5R + V8S + Y9K + V15K GFCWRVCSKRNGVRKCYRRCN — 441 Y5R + Y9K + V13A + V15K GFCWRVCVKRNGARKCYRRCN — 442 Y5R + Y9K + V15S + Y17S GFCWRVCVKRNGVRSCSRRCN — 443 Y5R + Y9R + V13A + V15K GFCWRVCVRRNGARKCYRRCN — 444 Y5R + Y9R + V13A + V15S GFCWRVCVRRNGARSCYRRCN — 445 Y5R + Y9R + V13L + V15K GFCWRVCVRRNGLRKCYRRCN — 446 Y5R + Y9R + V13L + Y17H GFCWRVCVRRNGLRVCHRRCN — 447 Y5R + Y9R + V15S + Y17S GFCWRVCVRRNGVRSCSRRCN — 448 Y5R + Y9R + Y17S + R19H GFCWRVCVRRNGVRVCSRHCN — 449 Y5R + Y9S + V13A + Y17S GFCWRVCVSRNGARVCSRRCN — 450 V6A + V8A + Y9K + V13L GFCWYACAKRNGLRVCYRRCN 98 451 V6A + Y9S + V13L + V15S GFCWYACVSRNGLRSCYRRCN — 452 V8A + R10S + V15S + Y17H GFCWYVCAYSNGVRSCHRRCN — 453 V8A + Y9R + V13L + V15S GFCWYVCARRNGLRSCYRRCN 92 454 Y9K + V13A + V15K + Y17H GFCWYVCVKRNGARKCHRRCN 90 455 W4A + Y5R + Y9K + V13A + V15K GFCARVCVKRNGARKCYRRCN — 456 W4A + Y5R + Y9K + V13A + Y17H GFCARVCVKRNGARVCHRRCN — 457 W4A + Y5R + Y9S + V13L + V15K GFCARVCVSRNGLRKCYRRCN — 458 W4A + Y9S + V13A + V15S + Y17H GFCAYVCVSRNGARSCHRRCN — 459 W4F + Y5H + V8A + Y9R + V15K GFCFHVCARRNGVRKCYRRCN — 460 W4F + Y5H + V8A + Y9R + Y17H GFCFHVCARRNGVRVCHRRCN — 461 W4F + Y5H + Y9K + V15K + Y17S GFCFHVCVKRNGVRKCSRRCN — 462 W4F + Y5K + V8S + V13L + V15K GFCFKVCSYRNGLRKCYRRCN — 463 W4F + Y5K + V8S + Y9K + V15K GFCFKVCSKRNGVRKCYRRCN — 464 W4F + Y5N + V8A + Y9R + Y17S GFCFNVCARRNGVRVCSRRCN — 465 W4F + Y5R + V13A + V15K + Y17S GFCFRVCVYRNGARKCSRRCN — 466 W4F + Y5R + V6A + V8A + Y17H GFCFRACAYRNGVRVCHRRCN — 467 W4F + Y5R + V6A + V8T + Y9S GFCFRACTSRNGVRVCYRRCN — 468 W4F + Y5R + V6A + Y9K + V13A GFCFRACVKRNGARVCYRRCN — 469 W4F + Y5R + V6A + Y9R + Y17H GFCFRACVRRNGVRVCHRRCN — 470 W4F + Y5R + V6A + Y9S + V15S GFCFRACVSRNGVRSCYRRCN — 471 W4F + Y5R + V6S + V8A + Y9R GFCFRSCARRNGVRVCYRRCN — 472 W4F + Y5R + V6S + V8A + Y9S GFCFRSCASRNGVRVCYRRCN — 473 W4F + Y5R + V8A + V13A + V15S GFCFRVCAYRNGARSCYRRCN — 474 W4F + Y5R + V8A + V13A + Y17H GFCFRVCAYRNGARVCHRRCN — 475 W4F + Y5R + V8A + V13A + Y17S GFCFRVCAYRNGARVCSRRCN — 476 W4F + Y5R + V8A + V15K + Y17H GFCFRVCAYRNGVRKCHRRCN — 477 W4F + Y5R + V8A + Y9K + V13A GFCFRVCAKRNGARVCYRRCN — 478 W4F + Y5R + V8A + Y9K + V15K GFCFRVCAKRNGVRKCYRRCN — 479 W4F + Y5R + V8A + Y9K + Y17H GFCFRVCAKRNGVRVCHRRCN — 480 W4F + Y5R + V8A + Y9S + V15K GFCFRVCASRNGVRKCYRRCN — 481 W4F + Y5R + V8S + V13A + V15S GFCFRVCSYRNGARSCYRRCN — 482 W4F + Y5R + V8S + V13L + V15K GFCFRVCSYRNGLRKCYRRCN — 483 W4F + Y5R + V8S + Y9K + V13A GFCFRVCSKRNGARVCYRRCN — 484 W4F + Y5R + V8S + Y9K + Y17H GFCFRVCSKRNGVRVCHRRCN — 485 W4F + Y5R + V8S + Y9R + V13L GFCFRVCSRRNGLRVCYRRCN — 486 W4F + Y5R + V8S + Y9S + V13A GFCFRVCSSRNGARVCYRRCN — 487 W4F + Y5R + V8S + Y9S + V15K GFCFRVCSSRNGVRKCYRRCN — 488 W4F + Y5R + Y9K + V13A + V15S GFCFRVCVKRNGARSCYRRCN — 489 W4F + Y5R + Y9R + V15K + Y17S GFCFRVCVRRNGVRKCSRRCN — 490 W4F + Y9S + V13A + V15K + Y17H GFCFYVCVSRNGARKCHRRCN — 491 W4G + Y5K + Y9K + V13L + V15K GFCGKVCVKRNGLRKCYRRCN — 492 W4G + Y5R + Gl2S + V13K + V15S GFCGRVCVYRNSKRSCYRRCN — 493 W4G + Y5R + R10K + V13K + V15A GFCGRVCVYKNGKRACYRRCN 77 494 W4S + V6S + V8A + V13S + V15S GFCSYSCAYRNGSRSCYRRCN — 495 W4S + Y5N + V6S + Y9K + Y17S GFCSNSCVKRNGVRVCSRRCN — 496 W4I + Y5N + V8A + Y9K + V13A GFCTNVCAKRNGARVCYRRCN — 497 Y5H + V6A + Y9R + V13A + V15S GFCWHACVRRNGARSCYRRCN — 498 Y5H + V6A + Y9R + V13L + V15K GFCWHACVRRNGLRKCYRRCN — 499 Y5H + V6A + Y9R + V13L + Y17H GFCWHACVRRNGLRVCHRRCN — 500 Y5H + V8A + R10K + V15A + Y17H GFCWHVCAYKNGVRACHRRCN — 501 Y5H + V8A + Y9R + V13L + Y17S GFCWHVCARRNGLRVCSRRCN — 502 Y5K + V6R + V13L + V15K + Y17H GFCWKRCVYRNGLRKCHRRCN — 503 Y5K + V6S + Y9R + V13L + V15S GFCWKSCVRRNGLRSCYRRCN — 504 Y5K + V8A + Y9R + V13A + V15S GFCWKVCARRNGARSCYRRCN 95 505 Y5K + V8S + Y9S + V13L + V15S GFCWKVCSSRNGLRSCYRRCN — 506 Y5K + Y9S + V13A + V15K + Y17S GFCWKVCVSRNGARKCSRRCN — 507 Y5N + V8A + Y9R + V13L + Y17H GFCWNVCARRNGLRVCHRRCN — 508 Y5N + Y9R + V13L + V15K + Y17H GFCWNVCVRRNGLRKCHRRCN — 509 Y5R + R10S + V13K + V15S + Y17H GFCWRVCVYSNGKRSCHRRCN 87 510 Y5R + V6A + R10K + G12S + V13K GFCWRACVYKNSKRVCYRRCN — 511 Y5R + V6A + R10K + V15A + Y17H GFCWRACVYKNGVRACHRRCN — 512 Y5R + V6A + V8A + V13K + V15A GFCWRACAYRNGKRACYRRCN — 513 Y5R + V6A + V8A + V13K + V15S GFCWRACAYRNGKRSCYRRCN — 514 Y5R + V6A + V8A + Y95 + Yl7H GFCWRACASRNGVRVCHRRCN — 515 Y5R + V6A + V8S + Y9R + V13L GFCWRACSRRNGLRVCYRRCN — 516 Y5R + V6A + Y9K + V13A + Y17S GFCWRACVKRNGARVCSRRCN — 517 Y5R + V6A + Y9K + V13L + V15S GFCWRACVKRNGLRSCYRRCN — 518 Y5R + V6A + Y9R + V13A + V15S GFCWRACVRRNGARSCYRRCN — 519 Y5R + V6A + Y9R + V13L + V15S GFCWRACVRRNGLRSCYRRCN — 520 Y5R + V6A + Y9S + V13A + V15S GFCWRACVSRNGARSCYRRCN — 521 Y5R + V6A + Y9S + V13L + Y17S GFCWRACVSRNGLRVCSRRCN — 522 Y5R + V6R + Y9R + V13A + V15S GFCWRRCVRRNGARSCYRRCN — 523 Y5R + V8A + R10K + V13K + V15S GFCWRVCAYKNGKRSCYRRCN 90 524 Y5R + V8A + Y9R + V13L + Y17H GFCWRVCARRNGLRVCHRRCN — 525 Y5R + V8A + Y9S + V13A + Y17S GFCWRVCASRNGARVCSRRCN — 526 Y5R + V8G + R10K + V15A + Y17H GFCWRVCGYKNGVRACHRRCN — 527 Y5R + V8G + V13K + V15A + Y17H GFCWRVCGYRNGKRACHRRCN — 528 Y5R + V8S + R10S + G12S + V13K GFCWRVCSYSNSKRVCYRRCN — 529 Y5R + V8S + Y9R + V13L + V15S GFCWRVCSRRNGLRSCYRRCN — 530 Y5R + Y9R + V13L + V15S + Y17S GFCWRVCVRRNGLRSCSRRCN — 531 Y5R + Y9S + V13A + V15K + Y17S GFCWRVCVSRNGARKCSRRCN — 532 Y5R + Y9S + V13L + V15S + Y17S GFCWRVCVSRNGLRSCSRRCN — 533 F2L + Y5R + V8A + R10S + V13K + V15A GLCWRVCAYSNGKRACYRRCN — 534 W4F + Y5N + Y9S + V13A + V15K + Y17H GFCFNVCVSRNGARKCHRRCN — 535 W4F + Y5R + V6A + V8A + Y9R + V15S GFCFRACARRNGVRSCYRRCN — 536 W4F + Y5R + V6A + V8S + Y9K + V13L GFCFRACSKRNGLRVCYRRCN — 537 W4F + Y5R + V6A + Y9K + V13L + V15K GFCFRACVKRNGLRKCYRRCN — 538 W4F + Y5R + V6A + Y9K + V13L + Y17H GFCFRACVKRNGLRVCHRRCN — 539 W4F + Y5R + V8A + Y9K + V13A + V15S GFCFRVCAKRNGARSCYRRCN — 540 W4F + Y5R + V8A + Y9S + V15K + Y17H GFCFRVCASRNGVRKCHRRCN — 541 W4F + Y5R + V8S + Y9K + V13A + V15K GFCFRVCSKRNGARKCYRRCN — 542 W4G + Y5H + V6R + V8S + Y9R + V15K GFCGHRCSRRNGVRKCYRRCN — 543 W45 + Y5H + V6R + V8S + G12S + V15A GFCSHRCSYRNSVRACYRRCN — 544 W45 + Y5R + V8S + V13S + V15A + Yl7H GFCSRVCSYRNGSRACHRRCN — 545 Y5R + V6A + V8A + Y9S + V13L + Y17S GFCWRACASRNGLRVCSRRCN — 546 W45 + Y5N + V6R + V8H + R10S + V13S + GFCSNRCHYSNGSRACHRRCN — V15A + Y17H 547 Y5G + V6A + C7V + V8N + R101 + N11S + GFCWGAVNYTSNCRACKRRCN — Gl2N + V13C + V15A + Y17K 548 F2S + Y5G + V6A + C7V + V8N + R101 + GSCWGAVNYTSNCRACKRRCN — N11S + Gl2N + V13C + V15A + Y17K The symbol “—” means “not analyzed”.

Example 2 Efficacy of NZ17074 Against Escherichia coli AID#172 in the Neutropenic Murine Peritonitis/Sepsis Model and Estimation of ED50 Introduction

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic peritonitis model. Treatment with 40 mg/kg meropenem was included as a positive control group. The colony counts in blood and peritoneal fluid were determined at 5 hours after treatment.

The murine peritonitis/sepsis model is a well-recognized model for studies of antimicrobial activity as described by N. Frimodt-Møller and J. D. Knudsen in Handbook of Animal Models of Infection (1999), ed. by O. Zak & M. A. Sande, Academic Press, San Diego, US.

Materials and Methods

-   -   30 outbred, NMRI female mice, 25-30 grams (Harlan Scandinavia)     -   E. coli AID#172 from Statens Serum Institute, Copenhagen,         Denmark. Clinical isolate from a human wound from 2003.         Multiresistant (Ampicillin, Ceftazidime, Aztreonam, Gentamicin,         Ciprofloxacin)     -   NZ17074 in Ringer Acetate, pH 6: 1.2 mg/ml, 6.0 ml. The solution         was stored at 4° C. until use. Analyses of the dose formulations         used were performed after completion of the in-life phase of the         study and gave the following results:

Intended concentration Measured concentration 1.2 mg/ml 1.11 mg/ml 0.6 mg/ml 0.51 mg/ml 0.3 mg/ml 0.24 mg/ml 0.15 mg/ml 0.14 mg/ml 0.075 mg/ml 0.043 mg/ml 0.03 mg/ml 0.010 mg/ml 0.016 mg/ml 0.002 mg/ml

-   -   Vehicle (Ringer Acetate pH 6). The solution was stored at 4° C.         until use     -   MERONEM® (AstraZeneca, 500 mg infusion substance, meropenem).         Lot no. 09466C     -   Date of expire: August 2013     -   Water, sterile     -   0.9% saline, sterile     -   Cyclophosphamide, APODAN® (A-Pharma, 1 g) Batch nr. 928491 Date         of expire: May 2012     -   5% Horse Blood Agar plates     -   Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m-6 a.m.

The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 3 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage. Mice were weighed the day before dosing.

Preparation of NZ17074 Solutions

The solution of 1.2 mg/ml was further diluted in PBS vehicle as follows:

0.6 mg/ml˜7.5 mg/kg: 1.5 ml of 1.2 mg/ml NZ17074+1.5 ml vehicle 0.3 mg/ml˜5.0 mg/kg: 1.5 ml of 0.6 mg/ml NZ17074+1.5 ml vehicle 0.15 mg/ml˜2.5 mg/kg: 1.5 ml of 0.3 mg/ml NZ17074+1.5 ml vehicle 0.075 mg/ml˜1.25 mg/kg: 1.5 ml of 0.15 mg/ml NZ17074+1.5 ml vehicle 0.03 mg/ml˜0.63 mg/kg: 1.5 ml of 0.075 mg/ml NZ17074+2.25 ml vehicle 0.016 mg/ml˜0.16 mg/kg: 1.5 ml of 0.03 mg/ml NZ17074+1.5 ml vehicle

Preparation Meropenem Solution

Treatment with meropenem 40 mg/kg was included as a positive control group. A total of 500 mg meropenem (one ampoule) was dissolved in 10 ml water ˜50 mg/ml This stock solution was further diluted to 4 mg/ml (0.4 ml 50 mg/ml+4.6 ml saline).

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule APODAN® (A-Pharma, 1 g) 1 g) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml 20 mg/ml+13.5 ml saline) for use on day −4 or to 5 mg/kg (8.25 ml 20 mg/ml+21.75 ml saline)) for use on day −1.

Treatment of Mice with Cyclophosphamide

The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁶CFU/ml. One hour before start of treatment (time −1 hr) mice were inoculated intraperitoneally with 0.5 ml of the E. coli suspension in the lateral lower quadrant of the abdomen. Approximately ½-1 hour after treatment, mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml corresponding to 30 mg/kg) as a pain relief.

Treatment of Mice

The mice were treated i.v. in the lateral tail vein over approximately 30 seconds with 10 ml/kg with a single dose of NZ17074, meropenem or vehicle at time 0 hour (see Table 1). The dosing was based on a mean weight of 30 g. Mice that weighed 28-32 g received 0.30 ml solution. Mice that weighed 27-28 g received 0.25 ml solution and mice that weighed 32.1-36 g received 0.35 ml solution.

TABLE 1 Treatment and sampling schedule in the murine peritonitis model. Inoculation Sampling and mouse no. i.p. at − 1 hr Intravenous treatment at 0 hr 0 hour 5 hours 0.5 ml of Vehicle, Ringer acetate 1-2-3 E. coli NZ17074 0.16 mg/kg 4-5-6 AID#172 NZ17074 0.30 mg/kg 7-8-9 1 × 10⁶ NZ17074 0.75 mg/kg 10-11-12 CFU/ml NZ17074 1.5 mg/kg 13-14-15 NZ17074 3.0 mg/kg 16-17-18 NZ17074 6.0 mg/kg 19-20-21 NZ17074 12 mg/kg 22-23-24 meropenem 40 mg/kg 25-26-27 No treatment 28-29-30 T indicates the time in relation to treatment. Numbers in the sampling columns are mouse identification numbers.

Clinical Scoring of Mice

The mice were observed during the study and scored 0-5 based on their behaviour and clinical signs.

-   -   Score 0: Healthy.     -   Score 1: Minor clinical signs of infection and inflammation e.g.         observations of minor signs of distress or changed activity.     -   Score 2: Clear signs of infection like, social withdrawal, lack         of curiosity, changed body position, piloerection, or changes in         pattern of movement.     -   Score 3: Severe signs of infection like stiff movements, lack of         curiosity, changed body position, piloerection, pain, or changes         in pattern of movement.     -   Score 4: Severe pain and the mouse was sacrificed immediately to         minimize the suffering of the animal.     -   Score 5: The mouse was dead.

Sampling

Colony counts were determined from blood and peritoneal fluid at 0 and 5 hours. The mice were anaesthetized with CO₂+O₂ and blood was collected from axillary cutdown in 1.5 ml EDTA coated eppendorf tubes. The mice were sacrificed immediately after blood sampling and a total of 2 ml sterile saline was injected i.p. and the abdomen gently massaged before it was opened and fluid sampled with a pipette. Each sample was then 10 fold diluted in saline and 20-microliter spots were applied on blue agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts were performed at the start of treatment and 5 hours after treatment. The CFU counts and the clinical score of the mice are shown in Table 3. The CFU numbers are log₁₀ transformed before performing calculations.

The CFU/ml in the inoculum was determined to 6.29 log₁₀. At start of treatment the mean log₁₀ CFU/ml in peritoneal fluid was 5.76 and in blood 5.13 and the CFU levels remained at a similar level in the vehicle group (5.72 and 4.65 log₁₀ CFU/ml in the peritoneum and blood respectively) at 5 hours after treatment. Slightly lower CFU levels were observed in blood and peritoneal fluid after treatment with NZ17074 0.16-3.0 mg/kg. Treatment with 6 and 12 mg/kg NZ17074 resulted in CFU levels significantly lower (p<0.001) than after vehicle treatment both in peritoneal fluid and in blood (Table 3). Also the meropenem treatment, 40 mg/kg, resulted in significant reduction compared to the vehicle treated mice both in blood (p<0.05) and peritoneal fluid (p<0.01).

The dose-response curves (data not shown) were calculated in GraphPad Prism using Sigmoidal dose-response (variable slope). From these ED50 values were determined to 3.09±2.07 mg/kg in peritoneal fluid and 3.17±0.53 mg/kg in blood.

The maximum effect of NZ17074, E_(max), was defined as the log CFU difference between no response and maximum response. No response was characterised as colony counts at the same level as determined for vehicle treated mice. The E_(max) was calculated as the difference between the “Top plateau” and “Bottom plateau” in GraphPad Prism using Sigmoidal dose-response to be 4.72 log₁₀ CFU for the peritoneal fluid and 3.15 log₁₀ CFU for the blood.

In addition the 1, 2 and 3 log killing, defined as the dose required to obtain 1, 2 or 3 log reduction in bacterial loads compared to start of treatment, were estimated using GraphPad Prism. The 1, 2 and 3 log killing of NZ17074 was 1.11 mg/kg, 2.95 mg/kg and 4.73 mg/kg respectively in peritoneal fluid and 0.25 mg/kg, 2.75 mg/kg and 3.78 mg/kg respectively in blood.

No or only mild clinical score was observed in all of the treatment groups (Table 3).

Discussion and Conclusion

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic peritonitis/sepsis model.

The ED50 values for NZ17074 were determined to 3.09±2.07 mg/kg in the peritoneal fluid and 3.17±0.53 mg/kg in the blood. The 1 log killing was estimated to be 1.11 mg/kg in the peritoneal fluid and 0.25 mg/kg in the blood. The 2 log killing was estimated to be 2.95 mg/kg in the peritoneal fluid and 2.76 mg/kg in the blood. The 3 log killing was estimated to be 4.73 mg/kg in the peritoneal fluid and 3.78 mg/kg in the blood.

TABLE 2 Efficacy values for NZ17074 against E. coli AID#172 calculated in Graph Pad Prism. NZ17074 Peritoneal fluid Blood TOP 0.325 CFU/ml −0.985 CFU/ml BOTTOM −4.486 CFU/ml −4.138 CFU/ml Emax 4.811 CFU/ml 3.153 CFU/ml ED50 3.086 mg/kg 3.168 mg/kg R² 0.7524 0.6889 1 log killing 1.11 mg/kg 0.25 mg/kg 2 log killing 2.95 mg/kg 2.76 mg/kg 3 log killing 4.73 mg/kg 3.78 mg/kg

TABLE 3 Colony counts of E. coli AID#172 in blood and peritoneal fluid from neutropenic mice treated with a single dose of NZ17074, meropenem or vehicle. Clinical score log₁₀ CFU Treatment Mouse T = 0 T = 5 mean mean T = 0 hour no. Time hour hours PF PF Blood Blood Vehicle 1 T = 5 1 1 5.74 5.72 5.05 4.65 2 T = 5 1 0 5.54 4.78 3 T = 5 1 1 5.88 4.11 NZ17074 4 T = 5 1 1 5.16 5.31 4.27 4.54 0.16 mg/kg 5 T = 5 1 1 4.78 4.19 6 T = 5 1 0 5.98 5.18 NZ17074 7 T = 5 1 0 2.76 4.28 1.40 2.88 0.30 mg/kg 8 T = 5 1 1 5.74 4.63 9 T = 5 1 0 4.27 2.60 NZ17074 10 T = 5 1 0 5.74 5.16 5.07 4.46 0.75 mg/kg 11 T = 5 1 1 4.95 4.30 12 T = 5 1 1 4.78 4.00 NZ17074 13 T = 5 1 0 3.33 4.41 3.51 3.99 1.5 mg/kg 14 T = 5 1 1 4.72 3.92 15 T = 5 1 0 5.18 4.54 NZ17074 18 T = 5 1 1 4.74 3.91 3.86 2.81 3.0 mg/kg 17 T = 5 1 1 4.74 3.57 18 T = 5 1 0 2.24 1.00 NZ17074 19 T = 5 1 1 2.18 2.12*** 1.00 1.00*** 8.0 mg/kg 20 T = 5 1 1 2.18 1.00 21 T = 5 1 1 2.00 1.00 NZ17074 22 T = 5 1 1 1.00 1.36*** 1.00 1.00*** 12 mg/kg 23 T = 5 1 0 1.69 1.00 24 T = 5 1 0 1.40 1.00 Meropenem 25 T = 5 1 1 3.92 2.64** 2.48 2.38* 40 mg/kg 28 T = 5 1 1 1.70 1.70 27 T = 5 1 0 2.30 2.95 28 T = 0 1 5.84 5.78 5.08 None 29 T = 0 1 5.78 4.98 5.13 30 T = 0 1 5.65 5.34 Stars indicate significantly different from vehicle group (Anova; multiple comparison). *corresponds to p < 0.05; **corresponds to p < 0.01; ***corresponds to p < 0.001. Detection limit 1.4 log₁₀ CFU/ml. Samples with no detectable bacteria is presented as 1.0 log₁₀ CFU/ml.

Example 3 Peritonitis/Sepsis Model: Effect Over Time of 7.5 Mg/Kg NZ17074 Against Escherichia coli AID#172 in Neutropenic NMRI Mice Introduction

The purpose of this study was to investigate the in vivo efficacy of NZ17074 following intravenous (i.v.) administration of a single dose of 7.5 mg/kg. The effect was tested against Escherichia coli AID#172 in the peritonitis model in neutropenic NMRI mice to avoid the use of mucin as normally applied in the murine peritonitis model. The mice were rendered neutropenic by cyclophosphamide injections. Treatment with 40 mg/kg meropenem was included as a positive control group and treatment with vehicle was included as a negative control group. The colony counts in peritoneal fluid and blood were determined at 2 and 5 hours after treatment.

Materials and Methods

-   -   30 outbred, NMRI female mice, 28-32 grams (Harlan Scandinavia)     -   Escherichia coli AID#172 from Statens Serum Institute,         Copenhagen, Denmark. Clinical isolate from a human wound         from 2003. Multiresistant (Ampicillin, Ceftazidime, Aztreonam,         Gentamicin, Ciprofloxacin)     -   NZ17074 in Ringer Acetate pH 6, 1.2 ml 0.75 mg/ml. Analyses of         the dose formulation performed after the study showed a         concentration of approx. 0.78 mg/ml.     -   Vehicle (Ringer Acetate pH 6) 3 ml.     -   MERONEM® (AstraZeneca, 500 mg meropenem infusion substance). Lot         no. 09466C     -   Date of expiration: August 2013     -   APODAN® (A-Pharma, 1 g cyclophosphamide) Batch nr. 928491     -   Date of expiration: May 2012     -   Water, sterile     -   0.9% saline, sterile     -   5% Horse Blood Agar plates     -   Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m.-6 a.m. The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 3 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage.

NZ17074 Solution

A solution of 0.75 mg/ml of each test compound was stored at +4° C. until one hour before injection, thereafter at room temperature.

Preparation of Meropenem Solution

A total of 500 mg meropenem (one ampoule) was dissolved in 10 ml water˜50 mg/ml the day of use. This stock solution was further diluted to 4 mg/ml (0.4 ml 50 mg/ml+4.6 ml saline).

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule APODAN® (A-Pharma)) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml of 20 mg/ml+13.5 ml saline) for use on day −4 or to 5.5 mg/ml (8.25 ml of 20 mg/ml+21.75 ml saline) for use on day −1.

Treatment of Mice with Cyclophosphamide

The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁶CFU/ml.

One hour before start of treatment (time −1 hour) mice were inoculated intraperitoneally with 0.5 ml of the E. coli suspension in the lateral lower quadrant of the abdomen.

2.5 hours after treatment, when clinical signs of infection were significant, mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml, corresponding to 30 mg/kg) as a pain relief.

Scoring of Mice

The mice were clinically scored for signs of infection at the time of each sampling.

-   Score 0: Healthy. -   Score 1: Minor clinical signs of infection and inflammation e.g.     observations of minor signs of distress or changed activity. -   Score 2: Clear signs of infection like, social withdrawal, lack of     curiosity, changed body position, piloerection, or changes in     pattern of movement. -   Score 3: Severe signs of infection like stiff movements, lack of     curiosity, forced ventilation, changed body position, piloerection,     pain, or changes in pattern movement. -   Score 4: Severe pain and the mouse was sacrificed immediately to     minimize the suffering of the animal. -   Score 5: The mouse was dead.

Treatment of Mice

The mice were treated i.v. in the lateral tail vein over approximately 30 seconds with a single dose of NZ17074, meropenem or vehicle at time 0 hour (see Table 1). The dosing was based on a mean weight of 30 g. Mice that weighed 28-32 g received 0.30 ml solution. Mice that weighed 27-28 g received 0.25 ml solution and mice that weighed 32.1-36 g received 0.35 ml solution. Mouse 17 accidently received 0.35 ml although it weighed 29.5 g. This does not seem to have influenced the results as the CFU levels in this mouse was very similar to the other two mice in the group.

TABLE 4 Treatment and sampling schedule in the murine peritonitis model. Inoculation Treatment Sampling Sampling Sampling T = −1 hour T = 0 hour T = 0 hour T = 2 hours T = 5 hours 0.5 ml of NZ17074 4, 5, 6 E. coli Meropenem 7, 8, 9 AID#172 Vehicle 10, 11, 12 10⁶ CFU/ml (Ringer Acetate) NZ17074 16, 17, 18 Meropenem 19, 20, 21 Vehicle 22, 23, 24 (Ringer Acetate) None 25, 26, 27 None 28, 29, 30 T indicates the time in relation to treatment. Numbers in the sampling columns are mouse identification numbers.

Sampling

Colony counts were determined from blood and peritoneal fluid at 0, 2 and 5 hours after treatment according to Table 1.

The mice were anesthetized with O₂+CO₂ and blood was collected by axilliary cut down. The mice were sacrificed by cervical dislocation and a total of 2 ml sterile saline was injected i.p. and the abdomen gently massaged before it was opened and fluid sampled with a pipette. Each sample was 10 fold diluted in saline and 20-microliter spots were applied on blood agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts and the clinical scores of the mice are shown in Table 2. The CFU numbers are log₁₀ transformed before performing calculations to obtain a normal distribution.

The CFU/ml in the inoculum was determined to 6.30 log₁₀. At start of treatment the mean log₁₀ CFU/ml in the peritoneal fluid was 3.57 and in the blood 3.54 and the CFU level increased to 5.43 and 4.58 in the peritoneal fluid and the blood respectively after 2 hours in vehicle treated animals and to 5.72 and 4.74 in the peritoneal fluid and the blood respectively after 5 hours in vehicle treated mice, which was as expected.

At 2 hours after treatment with NZ17074 significantly lower CFU levels were observed both in the blood and the peritoneal fluid compared to the vehicle treatment (p<0.001).

A further reduction of the CFU levels was observed at 5 hours after treatment with NZ17074 both in the blood and in the peritoneal fluid (p<0.001 compared to vehicle control). The CFU levels were more the 3 log₁₀ CFU/ml lower than after vehicle treatment.

Also meropenem treatment resulted in significantly (p<0.01) reduced CFU levels compared to vehicle treatment in the peritoneal fluid at both 2 and 5 hours after treatment but in the blood only at 5 hours after treatment. The lack of significance in the blood at 2 hours after treatment may reflect the large variability in the vehicle group rather than poor effect of meropenem.

The difference in CFU levels after NZ17074 or meropenem treatment compared to vehicle treatment was:

NZ17074, 2 hours: peritoneum −1.63 blood −2.50 log cfu/ml 7.5 mg/kg log cfu/ml 5 hours: peritoneum −3.76 blood −3.74 log cfu/ml log cfu/ml Meropenem 2 hours: peritoneum −1.51 blood −0.82 log cfu/ml 40 mg/kg log cfu/ml 5 hours: peritoneum −1.51 blood −1.64 log cfu/ml log cfu/ml All mice had only mild or no symptoms of infection (Table 2).

Discussion and Conclusion

The purpose of this study was to investigate the efficacy of NZ17074 following intravenous (i.v.) administration of a single dose of 7.5 mg/kg in the neutropenic peritonitis model in NMRI mice. A significant (p<0.001) reduction of more the 3 log₁₀ CFU/ml compared to vehicle treatment was observed for NZ17074 in blood and peritoneal fluid at 5 hours after treatment. Also at 2 hours after treatment with NZ17074 a significant reduction (p<0.001) both in the blood and peritoneal fluid was observed. Meropenem showed a significant reduction compared to the vehicle group (p<0.01) both in the blood and in the peritoneal fluid at 5 hours but at 2 hours after treatment only in the peritoneal fluid.

TABLE 5 Colony counts of E. coli AID#172 in mice treated with a single dose of NZ17074, vehicle or meropenem Score log₁₀ CFU id Time of T = 0 T = 2 T = 5 mean Mean Treatment no. sampling hour hours hours PF PF Blood Blood NZ17074 4 T = 5 0 0 2.18 1.96*** 1.00 1.00*** 7.5 mg/kg 5 T = 5 0 1 2.30 1.00 6 T = 5 0 1 1.40 1.00 Meropenem 7 T = 5 0 0 4.38 4.21** 3.20 3.10** 40 mg/kg 8 T = 5 0 0 4.00 2.85 9 T = 5 0 0 4.26 3.26 10 T = 5 0 0 5.39 5.72 4.63 4.74 Vehicle 11 T = 5 0 0 5.99 5.24 12 T = 5 0 0 5.78 4.36 NZ17074 16 T = 2 0 1 4.24 3.79** 2.04 2.08** 7.5 mg/kg 17 T = 2 0 1 3.60 2.20 18 T = 2 0 1 3.54 2.00 Meropenem 19 T = 2 0 1 4.12 3.92** 3.60 3.76 40 mg/kg 20 T = 2 0 1 3.40 3.57 21 T = 2 0 1 4.24 4.11 Vehicle 22 T = 2 0 0 4.89 5.43 3.21 4.58 23 T = 2 0 1 5.65 5.39 24 T = 2 0 0 5.74 5.15 None 25 T = 2 0 0 4.45 5.08 4.39 4.33 26 T = 2 0 0 5.42 4.57 27 T = 2 0 0 5.38 4.02 None 28 T = 0 0 1.88 3.57 1.00 3.54 29 T = 0 0 3.71 4.27 30 T = 0 0 5.13 5.35 PF: peritoneal fluid. Used inoculum: 1.97 × 10⁶ CFU/ml. ^(#) Mouse received 0.35 ml instead of 0.30 ml of test compound *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle group.

Example 4 Neutropenic Thigh Infection Model: Efficacy of NZ17074 Against Escherichia coli AID#172 and Estimation of ED50 Introduction

The purpose of this study was to investigate the dose-response relationship following intravenous (i.v.) administration of a single dose of NZ17074 ranging from 0.16-12 mg/kg. The effect was tested against E. coli AID#172 in the neutropenic thigh model. Treatment with 40 mg/kg meropenem was included as a positive control group. The colony counts in thighs were determined at 5 hours after treatment.

The thigh infection model is a well-recognized model for studies of antimicrobial effect and tissue penetration as described by S. Gudmundsson & H. Erlensdóttir Handbook of Animal Models of Infection (1999), ed. by O. Zak & M. A. Sande, Academic Press, San Diego, US and in several publications. Reviewed by D. Andes & C. Craig: Animal model pharmacokinetics and pharmacodynamics: a critical review. International Journal of Antimicrobial Agents 19(4): 261-268.

Materials and Methods

-   -   40 outbred, NMRI female mice, 25-30 grams (Harlan Scandinavia)     -   E. coli AID#172 from Statens Serum Institute, Copenhagen,         Denmark: Clinical isolate from a human wound from 2003.         Multiresistant (Ampicillin, Ceftazidime, Aztreonam, Gentamicin,         Ciprofloxacin)     -   NZ17074 in Ringer Acetate, pH 6: 1.2 mg/ml, 6.0 ml. The solution         was stored at 4° C. until use. Analyses of the dose formulations         used were performed after completion of the in-life phase of the         study and gave the following results:

Intended concentration Measured concentration 1.2 mg/ml 1.11 mg/ml 0.6 mg/ml 0.51 mg/ml 0.3 mg/ml 0.24 mg/ml 0.15 mg/ml 0.14 mg/ml 0.075 mg/ml 0.043 mg/ml 0.03 mg/ml 0.010 mg/ml 0.016 mg/ml 0.002 mg/ml

-   -   Vehicle (Ringer Acetate pH 6). The solution was stored at 4° C.         until use     -   MERONEM® (AstraZeneca, 500 mg infusion substance, meropenem).         Lot no. 09466C     -   Date of expire: August 2013     -   Water, sterile     -   0.9% saline, sterile     -   SENDOXAN® (Cyclophosphamide, Baxter, 1 g) Batch nr. 0A671C Date         of expire: January 2013     -   5% Horse Blood Agar plates     -   Lactose bromthymol blue agar plates

Laboratory Animal Facilities and Housing of Mice

The temperature and humidity were registered daily in the animal facilities. The temperature was 21+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 10-20 times, and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m-6 a.m.

The mice had free access to domestic quality drinking water and food (2016, Harlan). The mice were housed in Type 3 macrolone cages with 4 mice/cage. The bedding was Aspen Wood from Tapvei. Further the animals were offered paper strands from Sizzle-nest as nesting material. Mice were marked on the tail for individual identification within the cage. Mice were weighed the day before dosing.

Preparation of NZ17074 Solutions

The solution of 1.2 mg/ml was further diluted in PBS vehicle as follows:

0.6 mg/ml˜7.5 mg/kg: 1.5 ml of 1.2 mg/ml NZ17074+1.5 ml vehicle 0.3 mg/ml˜5.0 mg/kg: 1.5 ml of 0.6 mg/ml NZ17074+1.5 ml vehicle 0.15 mg/ml˜2.5 mg/kg: 1.5 ml of 0.3 mg/ml NZ17074+1.5 ml vehicle 0.075 mg/ml˜1.25 mg/kg: 1.5 ml of 0.15 mg/ml NZ17074+1.5 ml vehicle 0.03 mg/ml˜0.63 mg/kg: 1.5 ml of 0.075 mg/ml NZ17074+2.25 ml vehicle 0.016 mg/ml˜0.16 mg/kg: 1.5 ml of 0.03 mg/ml NZ17074+1.5 ml vehicle

Preparation Meropenem Solution

Treatment with meropenem 40 mg/kg was included as a positive control group.

A total of 500 mg meropenem (one ampoule) was dissolved in 10 ml water˜50 mg/ml This stock solution was further diluted to 4 mg/ml (0.4 ml 50 mg/ml+4.6 ml saline).

Preparation of Cyclophosphamide

A total of 1 g cyclophosphamide (one ampoule SENDOXAN® (Cyclosphosphamide, Baxter, 1 g) was dissolved in 50 ml water˜20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml 20 mg/ml+13.5 ml saline) for use on day −4 or to 5 mg/kg (8.25 ml 20 mg/ml+21.75 ml saline)) for use on day −1.

Treatment of Mice with Cyclophosphamide

The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (200 mg/kg) and 1 day (100 mg/kg) prior to inoculation.

Inoculation of Mice

Fresh overnight E. coli AID#172 colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately 2×10⁷ CFU/ml. One hour before start of treatment (time −1 hour) mice were inoculated intramuscularly with 0.05 ml of the E. coli suspension in the left hind leg. Approximately ½ hour before inoculation mice were treated orally with 45 microliters neurophen (20 mg ibuprofen/ml corresponding to 30 mg/kg) as a pain relief.

Treatment of Mice

The mice were treated i.v. in the lateral tail vein over approximately 30 seconds with 10 ml/kg with a single dose of NZ17074, meropenem or vehicle at time 0 hour (see Table 1). The dosing was based on a mean weight of 30 g. Mice that weighed 28-32 g received 0.30 ml solution. Mice that weighed 27-28 g received 0.25 ml solution and mice that weighed 32.1-36 g received 0.35 ml solution.

TABLE 6 Treatment and sampling schedule in the murine thigh model. Inoculation Sampling and mouse no. i.m. at − 1 hr Intravenous treatment at 0 hr 0 hr 5 hours 0.05 ml of Vehicle, Ringer acetate 1-2-3-4 E. coli NZ17074 0.16 mg/kg 5-6-7-8 AID#172 NZ17074 0.30 mg/kg 9-10-11-12 2 × 10⁷ NZ17074 0.75 mg/kg 13-14-15-16 CFU/ml NZ17074 1.5 mg/kg 17-18-19-20 NZ17074 3.0 mg/kg 21-22-23-24 NZ17074 6.0 mg/kg 25-26-27-28 NZ17074 12 mg/kg 29-30-31-32 meropenem 40 mg/kg 33-34-35-36 No treatment 37-38-39-40 T indicates the time in relation to treatment. Numbers in the sampling columns are mouse identification numbers.

Clinical Scoring of Mice

The mice were observed during the study and scored 0-5 based on their behaviour and clinical signs.

-   Score 0: Healthy. -   Score 1: Minor clinical signs of infection and inflammation e.g.     observations of minor signs of distress or changed activity. -   Score 2: Clear signs of infection like, social withdrawal, lack of     curiosity, changed body position, piloerection, or changes in     pattern of movement. -   Score 3: Severe signs of infection like stiff movements, lack of     curiosity, changed body position, piloerection, pain, or changes in     pattern of movement. -   Score 4: Severe pain and the mouse was sacrificed immediately to     minimize the suffering of the animal. -   Score 5: The mouse was dead.

Sampling

Colony counts were determined from thighs at 0 and 5 hours. The mice were anaesthetized with CO₂+O₂ and sacrificed. Immediately after, skin was removed and the left hind leg was collected and frozen at −70° C. After thawing, the thighs were homogenized using a Dispomix® Drive. Each sample was then 10 fold diluted in saline and 20-microliter spots were applied on blue agar plates. All agar plates were incubated 18-22 hours at 35° C. in ambient air.

Results

The colony counts were performed at the start of treatment and 5 hours after treatment.

The CFU counts are shown in Table 3. The CFU numbers are log₁₀ transformed before performing calculations.

The CFU/ml in the inoculum was determined to 7.35 log₁₀ corresponding to 6.05 log₁₀ CFU/mouse. The high variability observed may be caused by suboptimal inoculation of some mice and resulting in too low CFU values. The lowest value in each group was therefore excluded from graphs and calculations (se Table 3). At start of treatment the mean log₁₀ CFU/ml was 4.93 and increased to 6.49 log₁₀ CFU/ml in the vehicle group at 5 hrs after treatment. Slightly lower CFU levels were observed after treatment with NZ17074 0.16-3.0 mg/kg. Significantly lower CFU levels were observed after treatment with 6 mg/kg (p<0.05) and 12 mg/kg (p<0.01) NZ17074 compared to vehicle treatment (Table 3). Meropenem treatment, 40 mg/kg, resulted in slight but not significant reduction compared to the vehicle treated mice.

The dose-response curves (not shown) were calculated in GraphPad Prism using Sigmoidal dose-response (variable slope). From this the ED50 value was determined to 5.9 mg/kg. However, a bottom plateau was not obtained and this value may therefore be underestimated.

The maximum effect of NZ17074, E_(max), was defined as the log CFU difference between no response and maximum response. No response was characterised as colony counts at the same level as determined for vehicle treated mice. The E_(max) was calculated as the difference between the “Top plateau” and “Bottom plateau” in GraphPad Prism using Sigmoidal dose-response to be 2.4 Δlog₁₀ CFU/ml. In addition the 1 log killing, defined as the dose required to obtain 1 log reduction in bacterial loads compared to start of treatment, was estimated using GraphPad Prism to 6.1 mg/kg. A 2 and 3 log killing was not obtained.

No clinical signs of infection were observed at any time point in any of the mice.

TABLE 7 Efficacy values for NZ17074 against E. coli AID#172 calculated in Graph Pad Prism TOP 1.1 Dlog₁₀ CFU/ml BOTTOM −1.3 Dlog₁₀ CFU/ml  Emax 2.4 Dlog₁₀ CFU/ml ED50 5.9 mg/kg R² 0.46 1 log killing 6.1 mg/kg

TABLE 8 Colony counts of E. coli AID#172 in thighs from neutropenic mice treated with a single dose of NZ17074, meropenem or vehicle. log₁₀ log₁₀ Treatment mouse Time of CFU Treatment mouse Time of CFU T = 0 hour no. sampling thigh mean T = 0 hrs no. sampling thigh mean Vehicle 1 T = 5 5.16¤ 6.49 NZ17074 21 T = 5 5.30 6.07 2 T = 5 6.47 3.0 mg/kg 22 T = 5 6.03 3 T = 5 6.13 23 T = 5 4.85¤ 4 T = 5 6.86 24 T = 5 6.89 NZ17074 5 T = 5 3.18¤ 5.16 NZ17074 25 T = 5 2.75 4.10* 0.16 mg/kg 6 T = 5 6.03 6.0 mg/kg 26 T = 5 4.54 7 T = 5 3.30 27 T = 5 1.48¤ 8 T = 5 6.15 28 T = 5 5.01 NZ17074 9 T = 5 2.00¤ 5.09 NZ17074 29 T = 5 2.48¤ 3.32** 0.30 mg/kg 10 T = 5 5.40 12 mg/kg 30 T = 5 3.27 11 T = 5 3.10 31 T = 5 3.19 12 T = 5 6.78 32 T = 5 3.51 NZ17074 13 T = 5 2.9¤ 6.33 Meropenem 33 T = 5 3.08¤ 4.25 0.75 mg/kg 14 T = 5 5.72 40 mg/kg 34 T = 5 3.81 15 T = 5 7.27 35 T = 5 4.99 16 T = 5 6.00 38 T = 5 3.94 NZ17074 17 T = 5 2.56¤ 5.62 None 37 T = 0 4.98 4.93 1.5 mg/kg 18 T = 5 6.23 38 T = 0 3.81¤ 19 T = 5 4.93 39 T = 0 4.79 20 T = 5 5.70 40 T = 0 5.01 ¤This value was excluded from calculations as it was considered an outlier. Stars indicate significantly different from vehicle group (Annova; multiple comparison). *corresponds to p < 0.05; **corresponds to p < 0.01. Detection limit 1.4 log₁₀ CFU/ml.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. 

What is claimed is:
 1. An isolated variant of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising a substitution at one or more positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and 21 of the amino acid sequence of SEQ ID NO: 2; wherein the variant has antimicrobial activity, and the number of substitutions is 1-11, wherein when said variant comprises a substitution selected from G1R or G1K, said variant further comprises a substitution at positions 5 or
 17. 2. The variant of claim 1, wherein the number of substitutions are 1-10, 1-9 substitutions, 1-8 substitutions, 1-7 substitutions, 1-6 substitutions, 1-5 substitutions, 1-4 substitutions, 1-3 substitutions, or 1-2 substitutions.
 3. The variant of any claim 1, which further comprises one or more (several) substitutions selected from the group consisting of G1A, G1D, G1F, G1H, G1I, G1K, G1M, G1Q, G1R, G1S, G1T, G1V, G1W, G1Y, F2A, F2G, F2H, F2I, F2L, F2M, F2P, F2S, F2V, F2W, F2Y, C3L, W4A, W4E, W4F, W4G, W41, W4L, W4M, W4N, W4Q, W4S, W4T, W4V, W4Y, Y5E, Y5F, Y5G, Y5H, Y5K, Y5N, Y5R, Y5S, Y5W, V6A, V6C, V6E, V6F, V6G, V6H, V61, V6L, V6M, V6N, V6Q, V6R, V6S, V6T, V6W, V6Y, C7V, V8A, V8F, V8G, V8H, V81, V8L, V8N, V85, V8T, V8W, V8Y, Y9A, Y9D, Y9F, Y9G, Y9H, Y91, Y9K, Y9M, Y9Q, Y9R, Y9S, Y9T, Y9V, Y9W, R10K, R10P, R10S, R10T, N11A, N11G, N11H, N11Q, N11R, N115, N11Y, G12A, G12D, G12E, G12F, G12H, G12K, G12N, G12R, G125, G12Y, V13A, V13C, V13F, V13G, V13H, V13K, V13L, V13P, V13Q, V13R, V135, V13T, V13W, V13Y, V15A, V15C, V15F, V15G, V15H, V15I, V15K, V15L, V15M, V15N, V15P, V15Q, V15R, V155, V15T, V15W, V15Y, Y17C, Y17F, Y17G, Y17H, Y17I, Y17K, Y17L, Y17M, Y17N, Y17Q, Y17R, Y17S, Y17T, Y17V, Y17W, R19D, R19H, R19K, R19M, R19T, R19Y, N21A, N21C, N21F, N21G, N21H, N21I, N21K, N21L, N21M, N21P, N21Q, N21R, N21S, N21T, N21W, and N21Y.
 4. The variant of claim 1, which comprises a substitution at two of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and
 21. 5. The variant of claim 1, which comprises a substitution at three of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and
 21. 6. The variant of claim 1, which comprises a substitution at four of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, and
 21. 7. The variant of claim 1, which comprises a substitution at one or more (several) of positions 1, 2, 4, 5, 6, 8, 9, 12, 13, 15, 17, and
 21. 8. The variant of claim 1, which comprises the amino acid sequence of any of SEQ ID NO: 3-9, 13-25, 29-46, 50-55, 57, 59-60, 62, 65-66, 79-85, 88-91, 93-94, 96-116, 118-119, 126, 128-129, 138-160, 167, 178, 183, 196-197, 205, 212-214, 216-219, 338-344, 347-349, 352-353, 356-357, 361-362, 369-370, 383, 390, 392, 494, 500, and
 543. 9. The variant of claim 1, which comprises the amino acid sequence of any of SEQ ID NO: 13-25, 46, 57, 59-60, 66, 89, 94, 96-116, 118, 129, 138-160, 167, 178, 183, 196-197, 205, 212-214, 216-219, 338-344, 347-349, 352-353, 356-357, 361-362, 369-370, 383, 390, 392, 494, 500, and
 543. 10. An isolated polynucleotide encoding the variant of claim
 1. 11. A nucleic acid construct comprising the polynucleotide of claim
 10. 12. An expression vector comprising the polynucleotide of claim
 10. 13. A host cell comprising the polynucleotide of claim
 10. 14. A method of producing a variant of an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2, comprising: a) cultivating the host cell of claim 13 under conditions suitable for the expression of the variant; and b) recovering the variant.
 15. A method of treating a microbial infection in a subject in need thereof comprising administering an amount of the variant of claim 1 effective to treat said microbial infection.
 16. A composition comprising the variant of claim 1 and an antibiotic. 